Polynucleotide probe and primer for detecting beer-clouding lactic acid bacteria and method of detecting beer-clouding lactic acid bacteria

ABSTRACT

An object of the present invention is to provide probes and primers for detecting beer-spoilage lactic acid bacteria with accuracy. The probes and primers for detecting beer-spoilage lactic acid bacteria according to the present invention each comprises a nucleotide sequence consisting of at least 15 nucleotides that hybridizes with the nucleotide sequence of SEQ ID NO: 1 or the complementary sequence thereof.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates to a method of detecting lactic acid bacteria which cause beer turbidity or cloudiness and affect beer quality. The present invention also relates to a protein specific to lactic acid bacteria which cause beer turbidity or cloudiness and a polynucleotide encoding the protein.

[0003] 2. Description of the Background Art

[0004] Beer is a drink that has limited carbon sources, contains alcohol and carbon dioxide gas, presents low pH and anaerobic conditions, and further contains substances having antimicrobial activity derived from hops such as isohumulone, making it difficult for microbial contamination or microbial growth to occur. However, it is known that when, even under these conditions, beer is contaminated with a certain kind of lactic acid bacteria which belong to genus Lactobacillus or genus Pediococcus, the bacteria grow and cause beer turbidity or cloudiness to greatly affect the quality of beer. Typical examples of such lactic acid bacteria include L. brevis, P. damnosus and L. lindneri but some other lactic acid bacteria are also confirmed to have such activity. However, it is known that even among lactic acid bacteria that belong to the same species, some strains grow in beer to cause turbidity and cloudiness (hereinafter referred to as “beer-spoilage lactic acid bacteria”) while others don't grow (hereinafter referred to as “non-beer-spoilage lactic acid bacteria”). This inconsistency often occurs with strains of L. brevis and P. damnosus. Therefore, beer-spoilage lactic acid bacteria cannot be directly detected simply by distinguishing the species.

[0005] Methods for the detection and distinction of lactic acid bacteria that affect the quality of beer have been studied to date. In a typical method, DNA is extracted from lactic acid bacteria and the presence of a particular gene of beer-spoilage lactic acid bacteria (hereinafter referred to as a “marker”) is confirmed by Southern hybridization reaction or the like. In particular today, a method of distinguishing lactic acid bacteria by amplifying a DNA sequence by a PCR (polymerase chain reaction) method using an oligonucleotide as a primer (Japanese Patent Laid-Open Publication No. 141899/1994) is used to distinguish lactic acid bacteria. This method has advantages such that only a small amount of bacterial cells is required for distinction, the operation is simple, and a result can be obtained in a short time.

[0006] When this method is used for distinguishing beer-spoilage lactic acid bacterial, its success or failure is most influenced by a marker and a primer sequence constructed based on the marker. Namely, beer-spoilage lactic acid bacteria having a marker and a primer sequence constructed from the marker can easily be detected, but beer-spoilage lactic acid bacteria not having such sequences cannot be detected even if they are present. On the other hand, when non-beer-spoilage lactic acid bacteria having such sequences are present, they are mistakenly detected as beer-spoilage lactic acid bacteria.

[0007] In the conventional method for distinguishing beer-spoilage lactic acid bacteria by PCR, an attempt has been made to solve the abovementioned problem by using a 16S ribosomal RNA gene as a marker and constructing a primer based on this marker. The 16S ribosomal RNA gene is a gene essential for sustaining bacterial viability and is highly preservable, but it has a region where the DNA sequence can be different in different organic species, which is called a variable region. This variable region is widely used in classification of organic species, genealogical analysis of evolution, and the like, and similarly for lactic acid bacteria, this gene can be used as a marker to detect and distinguish the abovementioned L. brevis, P. damnosus, L. lindneri, and the like.

[0008] However, there are two problems with this method. First, it is highly probable that since the DNA sequence of the primer is associated with a gene which is not directly related to the beer-spoilage ability, beer-spoilage lactic acid bacteria having a certain mutation in this site cannot be detected, even if they are present. The variable region of the 16S ribosomal RNA gene is considered to be vulnerable to mutation and thus beer-spoilage lactic acid bacteria having mutation in a small region of the PCR primer may not be detected even if they are present.

[0009] The other problem is that this method, which is essentially for the distinction of organic species, cannot be applicable to distinguish beer-spoilage lactic acid bacteria from non-beer-spoilage lactic acid bacteria, particularly for L. brevis and P. damnosus, because among lactic acid bacteria that belong to the same species, some strains could be beer-spoilage lactic acid bacteria while others could be non-beer-spoilage lactic acid bacteria, as mentioned above.

[0010] Accordingly, there has been a need for a marker gene which detects beer-spoilage lactic acid bacteria more accurately than the 16S ribosomal RNA gene.

[0011] An example of the most desirable marker to detect beer-spoilage lactic acid bacteria more accurately than the 16S ribosomal RNA gene is firstly the very causative gene that renders lactic acid bacteria beer-spoilage ability. Further, the next preferable marker is a base sequence which is known to be located in the proximity to the gene that renders lactic acid bacteria beer-spoilage ability.

[0012] There have been several reports on genes which are considered to be important for beer-spoilage lactic acid bacteria to acquire the beer-spoilage ability. For example, there is a report on a method of constructing a probe for the distinction of beer-spoilage lactic acid bacteria from a plasmid which is known to grow in lactic acid bacteria which have become resistant to a high hop concentration by gradual acclimatization to a medium containing a high concentration of hops (Japanese Patent Publication No. 3057552).

[0013] However, this method has a problem that it does not necessarily reflect the primary difference between beer-spoilage lactic acid bacteria and non-beer-spoilage lactic acid bacteria since the lactic acid bacteria are treated forcefully to acquire the hop resistance.

[0014] There are reports on obtaining genes specific to beer-spoilage lactic acid bacteria. For example, as for genes derived from L. brevis, horA obtained as a hop resistance gene (Journal of the American Society of Brewing Chemistry, 55, 137-140, 1997) and hitA, which is considered to be a gene related to manganese intake (Federation of European Microbiological Societies and Netherlands Society for Microbiology, Abstract of the Sixth Lactic Acid Bacteria Symposium, September 1999), have been reported. Though not quite satisfactorily, these gene are considered to be effective as markers for determining beer-spoilage lactic acid bacteria for L. brevis; however, horA erroneously identifies non-beer-spoilage lactic acid bacteria as beer-spoilage lactic acid bacteria at a high frequency for L. brevis and neither of the genes can distinguish beer-spoilage lactic acid bacteria for P. damnosus. Therefore, there has been a need for a marker for the detection of beer-spoilage lactic acid bacteria which is widely applicable and has a high correlation with the spoilage ability as compared to these previously reported markers.

SUMMARY OF THE INVENTION

[0015] An object of the present invention is to provide a method of distinguishing beer-spoilage lactic acid bacteria with improved accuracy and a probe, a primer, a primer pair, and an antibody for use in implementing this method.

[0016] Further, another object of the present invention is to provide a protein specific to lactic acid bacteria having beer-spoilage ability, a method of producing said protein, a polynucleotide encoding said protein, a vector carrying said polynucleotide, and a host transformed by said vector.

[0017] The present inventor has now succeeded in obtaining a region (SEQ ID NO: 79) which contains a specific gene widely found in beer-spoilage lactic acid bacteria (Example 1). Further, the present inventor has found that beer-spoilage lactic acid bacteria can be distinguished at an extremely high probability when the distinction was carried out by a PCR method using a primer constructed based on this gene (Example 2).

[0018] A polynucleotide probe for detecting beer-spoilage lactic acid bacteria according to the present invention comprises a nucleotide sequence consisting of at least 15 nucleotides, which hybridizes with the nucleotide sequence of SEQ ID NO: 1 or the complementary sequence thereof.

[0019] A method of detecting beer-spoilage lactic acid bacteria according to the present invention comprises the steps of hybridizing a polynucleotide probe of the present invention with a polynucleotide in a sample and then detecting a hybridization complex.

[0020] A polynucleotide primer for use in the detection of beer-spoilage lactic acid bacteria by a nucleic acid amplification reaction according to the present invention comprises a nucleotide sequence consisting of at least 15 nucleotides, which hybridizes with the nucleotide sequence of SEQ ID NO: 1 or the complementary sequence thereof.

[0021] A primer pair according to the present invention comprises two kinds of primers of the present invention and can amplify a genomic sequence specific to beer-spoilage lactic acid bacteria by a nucleic acid amplification method.

[0022] A method for detecting beer-spoilage lactic acid bacteria according to the present invention comprises the steps of amplifying a polynucleotide in a sample by a nucleic acid amplification reaction using a primer pair of the present invention and then detecting the resulting amplified polynucleotide.

[0023] A protein according to the present invention comprises the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 5, or the amino acid sequence of SEQ ID NO: 7.

[0024] Further, a protein according to the present invention is a protein which is obtainable by the steps of culturing a host comprising a recombinant vector carrying a polynucleotide described below and recovering the protein that is an expression product of said polynucleotide.

[0025] A polynucleotide according to the present invention encodes the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 5, or the amino acid sequence of SEQ ID NO: 7.

[0026] A recombinant vector according to the present invention carries a polynucleotide of the present invention.

[0027] A transformed host according to the present invention comprises a recombinant vector of the present invention.

[0028] A method for producing a protein according to the present invention comprises the steps of culturing a host comprising a recombinant vector carrying a polynucleotide of the present invention and recovering the protein that is an expression product of said polynucleotide.

[0029] An antibody according to the present invention is against a protein according to the present invention.

[0030] A method of detecting beer-spoilage lactic acid bacteria according to the present invention comprises the steps of reacting an antibody according to the present invention with a sample and detecting the antigen-antibody reaction.

BRIEF DESCRIPTION OF THE DRAWINGS

[0031]FIG. 1 shows the positions of the open reading frames and the restriction sites in the gene region (SEQ ID NO: 79) specific to beer-spoilage lactic acid bacteria.

DETAILED DESCRIPTION OF THE INVENTION

[0032] Polynucleotide Probe

[0033] As shown in Example 1, the present inventor succeeded in obtaining a gene region specific to beer-spoilage lactic acid bacteria (SEQ ID NO: 79). The obtained region has 10 open reading frames (ORFs) (designated as ORF1 to ORF10; see FIG. 1). In particular, genes of ORF1 to ORF3 form a single operon, and presumably ORF3 is a gene for glycosyltransferase or dolichol phosphate mannose synthetase, and ORF3 is a gene for teichoic acid galactosyltransferase. Accordingly, this operon is considered to be involved in sugar chain synthesis in the cell wall. It has been reported that a particular sugar chain is present in the cell surface layer of beer-spoilage lactic acid bacteria by Yasui et al. (FERM Microbiology Letters, 133, 181-186, 1995) and Tsuchiya et al. (Journal of the American Society of Brewing Chemistry, 58, 89-93, 2000). Further, as shown later in the example, markedly high correlation with beer-spoilage ability was found when detection was carried out using these genes as a marker. Therefore, at least, these three genes are involved in the sugar chain synthesis on the cell surface layer and contribute to the growth of beer-spoilage lactic acid bacteria in beer. In other words, it is highly probable that they are at least a part of causative gene related to the beer-spoilage ability. Accordingly, this operon region is useful as a marker for distinguishing beer-spoilage ability and a polynucleotide probe based on the nucleotide sequence of this region according to the present invention can be used for detecting beer-spoilage lactic acid bacteria.

[0034] Further, the present inventor fully studied both sides of the operon region to find out up to which extent the region could be used as a marker for distinguishing beer-spoilage ability and obtained the following results (see Example 2).

[0035] (1) When regions ORF1 through ORF4, and ORF8 are each used as a marker, beer-spoilage lactic acid bacteria can be distinguished from non-beer-spoilage lactic acid bacteria at a markedly high frequency for lactic acid bacterial such as L. brevis and P. damnosus.

[0036] (2) When regions ORF9 and ORF10 are each used as a marker, beer-spoilage lactic acid bacteria can be distinguished from non-beer-spoilage lactic acid bacteria at a markedly high frequency for L. brevis but not for P. damnosus.

[0037] (3) Regions ORF5, ORF6 and ORF7 cannot be used for distinguishing beer-spoilage lactic acid bacteria from non-beer-spoilage lactic acid bacteria at least as far as the inventor studied. (However, ORF7 can possibly be used for distinguishing species of lactic acid bacteria.)

[0038] Accordingly, a probe according to the present invention can be preferably a probe comprising at least 15 contiguous nucleotides of the nucleotide sequence of SEQ ID NO: 1 or the complementary sequence thereof.

[0039] A polynucleotide probe according to the present invention can be preferably a probe comprising at least 15 contiguous nucleotides of the sequence from position 2818 to position 8056 of SEQ ID NO: 1 or the complementary sequence thereof (sequence extending from ORF1 through ORF4 and ORF8; see FIG. 1), more preferably a probe comprising at least 15 contiguous nucleotides of the sequence from position 4202 to position 7513 of SEQ ID NO: 1 or the complementary sequence thereof (sequence extending from ORF1 through ORF3; see FIG. 1), and most preferably a probe comprising at least 15 contiguous nucleotides of the nucleotide sequence of SEQ ID NO: 2 (ORF1), SEQ ID NO: 4 (ORF2), or SEQ ID NO: 6 (ORF3) or the complementary sequence thereof.

[0040] A probe comprising at least 15 contiguous nucleotides of any one of SEQ ID NO: 2 (ORF1), SEQ ID NO: 4 (ORF2), and SEQ ID NO: 6 (ORF3) has an advantage that beer-spoilage lactic acid bacteria can be detected without difficulty even if the beer-spoilage lactic acid bacteria having the same ORF sequences but in different order.

[0041] A probe according to the present invention can also be a probe which has at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% homology to the nucleotide sequence comprising 15 contiguous nucleotides of the nucleotide sequence of SEQ ID NO: 1 or the complementary sequence thereof, and hybridizes with a genomic sequence specific to beer-spoilage lactic acid bacteria.

[0042] Examples of the “genomic sequence specific to beer-spoilage lactic acid bacteria” include the nucleotide sequence of SEQ ID NO: 1 and a partial sequence thereof, the nucleotide sequence of SEQ ID NO: 2 and a partial sequence thereof, the nucleotide sequence of SEQ ID NO: 4 and a partial sequence thereof, and the nucleotide sequence of SEQ ID NO: 6 and a partial sequence thereof.

[0043] In the present invention, the term “hybridize” means to hybridize with a target nucleotide sequence under stringent conditions and not to hybridize with a nucleotide sequence other than the target nucleotide sequence. The stringent conditions can be determined depending on Tm (° C.) of the double strand of a probe sequence (or a primer sequence described below) and a complementary chain thereof, the necessary salt concentration, or the like, and it is a well-known technique to the skilled in the art to set appropriate stringent conditions after selecting a sequence for a probe (for example, see J. Sambrook, E. F. Frisch, T. Maniatis; Molecular Cloning, 2nd edition, Cold Spring Harbor Laboratory, 1988). For example, when hybridization is carried out using a probe consisting of 15 nucleotides under stringent conditions appropriate to this probe (at a temperature slightly lower than the Tm determined by a nucleotide sequence and at an appropriate salt concentration), the hybridization takes place specifically with a sequence complementary to this nucleotide sequence and not with a sequence non-complementary to this nucleotide.

[0044] In the present invention, the term “polynucleotide probe” means a probe that is used for means of detecting nucleic acid, such Southern hybridization, Northern hybridization, and colony hybridization.

[0045] In the present invention, the term “polynucleotide” includes DNA, RNA, and PNA (peptide nucleic acid).

[0046] A polynucleotide probe according to the present invention has at least 15 nucleotides length, more preferably at least 20 nucleotides length.

[0047] A polynucleotide probe according to the present invention can be prepared by chemical synthesis of nucleic acid according to an ordinary method such as a phosphite triester method (Hunkapiller, M. et al., Nature, 310, 105, 1984), or by obtaining the whole DNA of beer-spoilage lactic acid bacteria which belong to L. brevis and then appropriately obtaining a DNA fragment containing a target nucleotide sequence based on a nucleotide sequence disclosed in this specification using a PCR method or the like, according to the method described in the Example below.

[0048] Method for Detecting Beer-Spoilage Lactic Acid Bacteria Using Polynucleotide Probe

[0049] A detection method using a polynucleotide probe can be carried out by hybridizing a polynucleotide probe according to the present invention with a nucleic acid sample and then detecting a hybridization complex, namely a nucleotide double strand. In a detection method according to the present invention, the presence of the hybridization complex indicates the presence of beer-spoilage lactic acid bacteria.

[0050] In a detection method using a probe, the term “hybridize” means the same as described in the polynucleotide probe section above.

[0051] In a detection method using a probe, the probe can be labeled. Examples of the labels include those using radioactivity (e.g., ³²P, ¹⁴C and ³⁵S), fluorescence (e.g., FITC and europium), and enzyme reactions, for example, with chemical coloring (e.g., peroxidase and alkaline phosphatase).

[0052] Detection of a hybridization complex can be carried out using conventional techniques such as Southern hybridization and colony hybridization (for example, see J. Sambrook, E. F. Frisch, T. Maniatis; Molecular Cloning, 2nd edition, cold Spring Harbor Laboratory, 1989).

[0053] A test sample can be a sample suspected to contain beer-spoilage lactic acid bacteria and more specifically, a bacterial colony detected by a microbial examination of beer.

[0054] Primer and Primer Pair

[0055] A primer and a primer pair according to the present invention each can hybridize with a genomic sequence specific to beer-spoilage lactic acid bacteria. Accordingly, a primer pair according to the present invention can be used for detecting beer-spoilage lactic acid bacteria by a nucleic acid amplification method such as a PCR method.

[0056] A primer according to the present invention can preferably be a primer comprising at least 15 contiguous nucleotides of the nucleotide sequence of SEQ ID NO: 1 or the complementary sequence thereof.

[0057] In the present invention, the term “primer” means a nucleotide sequence for use in a nucleic acid amplification method such as a PCR method.

[0058] In the present invention, the term “primer pair” means a pair of primers for use in a nucleic acid amplification method such as a PCR method.

[0059] A primer according to the present invention can comprise at least 15 nucleotides (preferably 15 to 30 nucleotides), preferably at least 20 nucleotides (more preferably 20 to 30 nucleotides).

[0060] A primer pair according to the present invention can be selected so that a genomic sequence specific to beer-spoilage lactic acid bacteria can be amplified by a nucleic acid amplification method such as a PCR method. A nucleic acid amplification method is known and the selection of the primer pair in the nucleic acid amplification method will be understood by those skilled in the art. For example, in a PCR method, primers can be selected so that one of the two primers attaches to one chain of a double stranded DNA specific to beer-spoilage lactic acid bacteria, the other primer attaches to the other chain of the double stranded DNA and one primer attaches to an extended chain extended by the other primer.

[0061] More specifically, a primer pair can be selected so that one primer comprises at least 15 contiguous nucleotides of the nucleotide sequence of SEQ ID NO: 1 and the other primer comprises at least 15 contiguous nucleotides of the sequence complementary to the nucleotide sequence of SEQ ID NO: 1. Further, since particularly the regions of ORF1, ORF2, ORF3, ORF4, and ORF 8 are considered to be specific to beer-spoilage lactic acid bacteria, a primer can be designed so that these regions can be amplified (see Examples 2 and 3).

[0062] Further, a primer pair according to the present invention can be preferably a primer pair in which one primer comprises at least 15 contiguous nucleotides of the nucleotide sequence from position 2818 to position 8056 of SEQ ID NO: 1 (sequence covering ORF1 through ORF 4 and ORF8) and the other primer comprises at least 15 contiguous nucleotides of the sequence complementary to the nucleotide sequence from position 2818 to position 8056 of SEQ ID NO: 1, more preferably a primer pair in which one primer comprises at least 15 contiguous nucleotides of the nucleotide sequence from position 4202 to position 7513 of SEQ ID NO: 1 (sequence covering ORF1 through ORF 3) and the other primer comprises at least 15 contiguous nucleotides of the sequence complementary to the nucleotide sequence from position 4202 to position 7513 of SEQ ID NO: 1, and most preferably a primer pair in which one primer comprises at least 15 contiguous nucleotides of nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6 and the other primer comprises at least 15 contiguous nucleotides of the sequence complementary to the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6.

[0063] A primer pair which amplifies one of the sequences of SEQ ID NO: 2 (ORF1), SEQ ID NO: 4 (ORF2) and SEQ ID NO: 6 (ORF3) or a partial sequence thereof has an advantage that beer-spoilage lactic acid bacteria can be detected without difficulty even if the beer-spoilage lactic acid bacteria having the same ORF sequences but in different order.

[0064] A primer according to the present invention can also be a probe which has at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% homology to the nucleotide sequence comprising 15 contiguous nucleotides of the nucleotide sequence of SEQ ID NO: 1 or the complementary sequence thereof, and hybridizes with a genomic sequence specific to beer-spoilage lactic acid bacteria.

[0065] A primer according to the present invention can be chemically synthesized based on a nucleotide sequence disclosed in this specification by an ordinary method such as a phosphite triester method (Hunkapiller, M. et al., Nature, 310, 105, 1984). Primers can be prepared, for example, according to “Bio Experiment Illustrated 3, Virtually Amplifying PCR” (by Hiroki Nakayama, Shujun sha).

[0066] Method of Detecting Beer-Spoilage Lactic Acid Bacteria Using Primer Pair

[0067] The term “hybridize” in a detection method using a primer pair means the same as described in the polynucleotide probe section above.

[0068] As disclosed in the Example described below, the detection of beer-spoilage lactic acid bacteria using a primer pair can be carried out by obtaining DNA from a sample, performing PCR with a primer according to the present invention using this DNA as a template according to an ordinary method, and detecting the presence or absence of amplification of DNA fragments specific to beer-spoilage lactic acid bacteria. The PCR technique itself is well known (for example, see “Bio Experiment Illustrated 3, Virtually Amplifying PCR”) and those skilled in the art can carry out a method of the present invention using an appropriate primer. In a detection method using a primer pair according to the present invention, the presence of amplification products indicates the presence of beer-spoilage lactic acid bacteria.

[0069] A test sample can be a sample suspected to contain beer-spoilage lactic acid bacteria and more specifically, a bacterial colony detected by a microbial examination of beer.

[0070] Protein Specific to Beer-Spoilage Lactic Acid Bacteria and Polynucleotide Encoding the Protein

[0071] A protein according to the present invention can be an indicator for the presence of beer-spoilage lactic acid bacteria since it is specifically expressed in beer-spoilage lactic acid bacteria. Accordingly, a protein according to the present invention is useful, for example, in preparing an antibody according to the present invention as described below.

[0072] A protein according to the present invention include a protein comprising the amino acid sequence of SEQ ID NO: 3 which has one or more modifications and has glucosyltransferase or dolichol phosphate mannose synthetase activity, and a protein comprising the amino acid sequence of SEQ ID NO: 7 which has one or more modifications and has teichoic acid galactosyltransferase activity.

[0073] The term “modification” in this specification means substitutions, deletions, additions and insertions. The number of modifications can be, for example, one to several, more specifically one to six. When two or more modifications are present, the type of introduced modifications can be the same or different.

[0074] Further, a protein according to the present invention can be specified as a protein which is obtainable by the steps of culturing a host comprising a recombinant vector carrying a polynucleotide according to the present invention and collecting the protein that is an expression product of said polynucleotide.

[0075] A polynucleotide according to the present invention encodes a protein specific to beer-spoilage lactic acid bacteria and is thus useful for producing a protein according to the present invention using gene recombination technology, as described below.

[0076] A polynucleotide according to the present invention can be a chemically synthesized DNA or naturally occurring DNA (derived from chromosomes or plasmids).

[0077] In obtaining a polynucleotide according to the present invention, a DNA fragment having the sequence of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6 is obtainable, for example, by obtaining the whole DNA of beer-spoilage lactic acid bacteria which belong to L. brevis, appropriately constructing a primer based on a nucleotide sequence disclosed in this specification so as to obtain a DNA fragment containing the target nucleotide sequence, and then amplifying the DNA fragment by a PCR method, according to the method described in the Example below, or alternatively, a polynucleotide can be prepared by nucleic acid chemical synthesis according to an ordinary method such as the phosphite triester method (Hunkapiller, M. et al., Nature, 310, 105, 1984). DNA having a nucleotide sequence of a degenerate sequence of the base sequence of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6 is obtainable by nucleic acid chemical synthesis based on an amino acid sequence disclosed in this specification.

[0078] A DNA sequence thus obtained can be confirmed by analysis according to a Maxam Gilbert method (for example, described in Maxam, A. M. and W. Gilbert, Proc. Natl. Acad. Sci. USA, 74, 560, 1977) or a Sanger method (for example, described in Sanger, F. and A. R. Coulson, J. Mol. Biol., 94, 441, 1975; Sanger, F., S. Nicklen and A. R. Coulson, Proc. Natl. Acad. Sci. USA, 74, 5463, 1977).

[0079] Recombinant Vector and Transformant

[0080] A recombinant vector according to the present invention can be constructed by incorporating a polynucleotide according to the present invention into a vector which is replicable in a host and carries a detectable marker gene, using an ordinary genetic engineering technique.

[0081] A recombinant vector according to the present invention can be produced according to a technique conventionally used for vector construction. More specifically, when a microorganism Escherichia coli is used as a host, for example, plasmid pUC119 (Takara Shuzo Co. Ltd.) and phagemid pBluescript II (Stratagene) can be used. When a yeast is used as a host, plasmid pYES2 (Invitrogen Corp.) can be used. When a mammalian cell is used as a host, plasmids such as pRC/RSV and pSRC/CMV (Invitrogen Corp.), a vector containing self-replication origin derived from viruses, such as EB virus plasmid Prep4, and pCEP4 (Invitrogen Corp.), can be used. When an insect cell is used as a host, an insect virus such as baculovirus can be used. When DNA is incorporated into a virus such as baculovirus, a transfer vector containing a base sequence homologous to the genome of the virus to be used can be used. Examples of such a transfer vector include plasmids such as pBacPAK8 and pAcUW31 commercially available from Clontech. When a polynucleotide according to the present invention is inserted into a transfer vector and the transfer vector and a virus genome are simultaneously introduced into a host, homologous recombination takes place between the transfer vector and the virus genome, yielding a virus in which the polynucleotide of the present invention is incorporated into the genome.

[0082] A vector with which a protein according to the present invention can be expressed in a nost can be constructed by operably linking a polynucleotide according to the present invention to regulatory sequences (for example, a promoter sequence and a terminator sequence) operable in the host, and incorporating the product into a vector.

[0083] In the present specification, the expression “operably link” means that regulatory sequences are linked to a polynucleotide according to the present invention so that the expression takes place under the control of the regulatory sequences in a host into which the polynucleotide according to the present invention is introduced. Generally, a promoter can be linked to the upstream of the gene and a terminator can be linked to the downstream of the gene.

[0084] A promoter to be used is not particularly limited as long as it exhibits promoter activity in a host to be transformed. For example, an adenovirus (Ad) early or late promoter, a Rous sarcoma virus (RSV) promoter, a cytomegalovirus (CMV) promoter, a simian virus (SV40) early or late promoter, a mouse mammalian tumor virus (MMTV) promoter, a thymidine kinase (tk) gene promoter of herpes simplex virus (HSV) can be used when an animal cell or fission yeast is used as a host cell; a ADH1 or GAL1 promoter can be used for a budding yeast; and the baculovirus polyhedron promoter or Drosophila metallothionein promoter can be used for an insect cell.

[0085] When a vector already carrying a promoter that functions in a host is used, a promoter contained in the vector can be operably linked to a polynucleotide according to the present invention.

[0086] For example, in plasmids pRC/RSV, pRC/CMV and the like, a cloning site is located downstream of a promoter operable in an animal cell, and thus a protein according to the present invention can be expressed by inserting a polynucleotide according to the present invention into the cloning site and then introducing the plasmid into the animal cell. Since the SV40 autonomous replication origin (ori) is already incorporated into these plasmids, the number of copies of such plasmids in a cell greatly increases when these plasmids are introduced into a culture cell transformed with an ori(−) SV40 genome, such as a COS cell, which can result in mass expression of the polynucleotide according to the present invention incorporated into said plasmids. Further, plasmid pYES2 for budding yeast has the GAL1 promoter and thus a vector with which a protein according to the present invention can be massively expressed in a budding yeast, such as INVSc1 (Invitrogen Corp.), can be constructed by inserting a DNA according to the present invention downstream of the GALL promoter of this plasmid or a derivative thereof.

[0087] A recombinant vector according to the present invention can be further linked to a marker gene to select transformants.

[0088] Further, a polynucleotide encoding an amino acid sequence of another protein or a part thereof at the 5′ or 3′ site of the polynucleotide according to the present invention can be linked in frame to a recombinant vector according to the present invention directly or via a polynucleotide encoding an amino acid sequence corresponding to a cleavage site peculiar to a specific protease. The amino acid sequence of another protein or a part thereof may have a signal peptide for secretion at its N terminal and in such a case, ligation to the 5′ site is preferable.

[0089] A recombinant vector according to the present invention can be introduced into a host according to an ordinary method suitable for the host to be transformed.

[0090] For example, when E. coli is used as a host, a calcium chloride method or an electroporation method can be used as described in Molecular Cloning, J. Sambrook et al., Cold Spring Harbor (1989) and the like. When a yeast cell is used as a host, a vector can be introduced, for example, using a Yeast Transformation Kit (Invitrogen Corp.) according to the lithium method. When a mammalian cell, insect cell, or the like is used as a host, a calcium phosphate method, a DEAE dextran method, an electroporation method, a lipofection method, or the like can be used. When a virus is used as a vector, the virus genome can be introduced into a host by using any of the abovementioned ordinary method for gene introduction or by infecting a host with a virus particle carrying the virus genome.

[0091] Transformants can be selected by a method appropriate to the nature of a marker gene contained in an introduced recombinant vector according to the present invention.

[0092] For example, when the marker gene is a tolerance gene for a drug which exhibits cytotoxic activity, a cell into which a recombinant vector according to the present invention is introduced can be cultured using a medium supplemented with this drug. Examples of a combination of such drug resistance gene and selectable drug include a neomycin resistance gene and neomycin, a hygromycin resistance gene and hygromycin, and a blasticidin S resistance gene and blasticidin S. Further, when the marker gene is a gene complementing nutritional requirement of a host, a cell into which a recombinant vector according to the present invention is introduced can be cultured using a minimal medium without the corresponding nutrient. In order to obtain a transformant in which a gene according to the present invention is incorporated into the host chromosome, for example, a recombinant vector according to the present invention is linearized by digesting with a restriction enzyme or the like, after which the resulting fragment is introduced into the host cell by the above-mentioned method, the resulting cell is cultured generally for several weeks, and then the transformant of interest can be selected using a detection marker introduced as an indicator. For example, a recombinant vector according to the present invention having a selectable drug resistance gene as mentioned above as a marker gene is introduced into a host by the abovementioned method, and then cultivation by passage is carried out on a medium supplemented with the selectable drug for several weeks or longer, after which selectable drug resistance clones survived as a colony were sucked up by a pipette and purified, and thus the transformant in which the gene according to the present invention is incorporated into the host chromosome can be obtained. The transformant thus obtained can be stored frozen and revived for use when needed, so that the trouble of constructing the transformant can be avoided and the function of the transformant can be maintained consistently, which makes it advantageous as compared to a strain into which a gene is temporarily incorporated.

[0093] A protein according to the present invention can be produced by culturing a transformed host according to the present invention and collecting the protein according to the present invention from the resulting culture.

[0094] A protein missing one or more residues at the N terminal and/or C terminal ends due to processing or the like in a host can also be satisfactorily used as an antigen to obtain an antibody according to the present invention. Further, as mentioned in the section of recombinant vector and host, when a DNA encoding an amino acid sequence of another protein or a part thereof can be linked in frame to a recombinant vector according to the present invention at the 5′ site or 3′ site of the gene according to the present invention directly or via a DNA encoding an amino acid sequence corresponding to a cleavage site specific to a specified protease, a protein according to the present invention is expressed as a fusion protein with the other protein or the part thereof, which is also included in the protein of the present invention.

[0095] When a microorganism is used as a host, it can be cultured using any kind of medium appropriately containing carbon sources, nitrogen sources, organic salts, inorganic salts, and the like ordinarily used for cultivation of this microorganism. The cultivation is carried out according to an ordinary culture method for microorganisms, such as solid culture and liquid culture including culture with stirring (e.g., test tube shake culture, reciprocating shake culture, and jar fermenter culture) and static culture (e.g., tank culture). The culture temperature can be appropriately selected within a range suitable for the microorganisms. For example, cultivation can be carried out in a medium at pH about 6 to about 8 at a culture temperature of about 15° C. to about 40° C. The cultivation time can be determined according to the culture conditions; however, it can generally be about 1 day to about 5 days.

[0096] When an animal cell is used as a host, it can be cultured using a medium ordinarily used for this animal cell. For example, cultivation can be carried out at 37° C. using a medium such as a DMEM medium supplemented with 10% v/v FBS in the presence of 5% v/v CO₂, changing the medium every several days. When the cells are grown to be confluent, they are dispersed into individual cells using an about 0.25% w/v trypsin PBS solution, after diluting several times, the culture is inoculated into a fresh culture vessel and the cultivation is continued until the cells grow up to a targeted amount to recover the cells. By carrying out passage culture in this way, the scale of the culture can be expanded to a desired size.

[0097] Similarly, when an insect cell is used as a host, cells can be obtained by passage culture, for example, at ₂5° C. to 35° C. in a medium for insect cells, such as Grace's medium supplemented with 10% v/v FBS and 2% w/v yeastolate. However, when cells that are easy to come off from a culture vessel, such as an Sf21 cell, are used as hosts, the passage can be preferably carried out by dispersing the cells by pipetting but not with a trypsin solution. Further, when a transformed cell containing a virus vector such as baculovirus vector is used, it is preferable to terminate the cultivation before grown cells die off due to cytoplasmic effect, for example, 72 hours after the start of cultivation.

[0098] After cultivation, a protein according to the present invention can be purified and isolated using any known isolation and purification procedure. For example, when a protein according to the present invention is produced intracellularly, this protein can be purified by the steps of collecting cells by centrifugation or the like after cultivation, suspending the cells in an ordinary buffer such as a buffer solution comprising 20 mM HEPES, pH 7, 1 mM EDTA, 1 mM DTT, and 0.5 mM PMSF to subsequently disrupt the cells using a polytron, ultrasonicator, Downs homogenizer, or the like, and recovering the supernatant fraction by ultra-centrifuging the resulting suspension at dozens of thousands ×g for several score minutes to about 1 hour to obtain a fraction containing the target protein according to the present invention.

[0099] Further, when a protein according to the present invention is secreted in a medium, a fraction containing the protein according to the present invention can be obtained as a supernatant fraction by centrifugation after cultivation. The supernatant fraction thus obtained is purified using an appropriate combination of purification procedures such as salting out, solvent precipitation, dialysis, ultrafiltration, gel electrophoresis, ion exchange chromatography, gel filtration chromatography, reverse chromatography, and affinity chromatography to recover the purified protein according to the present invention.

[0100] Antibody and Method of Detecting Beer-Spoilage Lactic Acid Bacteria Using the Antibody

[0101] An antibody according to the present invention can recognize a protein specific to beer-spoilage lactic acid bacteria. Accordingly, an antibody according to the present invention can be used for detecting beer-spoilage lactic acid bacteria by antibody-antigen reaction.

[0102] An antibody according to the present invention includes both polyclonal antibody and monoclonal antibody. Since the technology for antibody construction is well known, anyone skilled in the art can readily prepare either polyclonal or monoclonal antibody by an ordinary method using a protein according to the present invention as an immunogen.

[0103] For example, an antibody according to the present invention can be obtained by administering a protein according to the present invention as an antigen with an adjuvant or the like to mammals, birds, or the like generally used for antibody production, including rabbits, mice, rats, goats, sheep, chickens, hamsters, horses, and guinea-pigs, and then obtaining an antiserum from the animals. The antiserum can be used as it is, or if necessary, it can be further fractionated and purified as described below to obtain a polyclonal antibody. For monoclonal antibody production, for example, a mouse is used as the abovementioned mammal, the spleen of the immunized mouse is extracted, cell fusion is carried out between a lymphocyte prepared from this spleen and a mouse myeloma cell (for example, p3×63 6.5.3 ATCC No. CRL-1580) using polyethylene glycol 1500 (Behringer), and then the resulting fusion cells were screened for positive strains by using a limit dilution method to obtain a hybridoma which produces the target antibody (see C. Milstein & G. Kohler, Nature, 256, 497, 1975). Further, an antibody molecule expressed by means of genetic engineering can be obtained by cloning an antibody gene or a part thereof from the hybridoma cell expressing the antibody.

[0104] An antibody is purified from a material containing the antibody thus obtained, namely an antiserum or a culture supernatant of hybridoma cells, by a purification procedure comprising a combination of one or more steps generally used for protein purification (for example, affinity chromatography such as protein A affinity chromatography, protein G affinity chromatography, Avid gel chromatography, and anti-immunoglobulin immobilized gel chromatography, cation-exchange chromatography, anion-exchange chromatography, lectin affinity chromatography, pigment adsorption chromatography, hydrophobic alternate chromatography, gel permeation chromatography, reverse-phase chromatography, hydroxyapatite chromatography, fluoroapatite chromatography, metal chelating chromatography, isoelectric point chromatography, preparatory electrophoresis, and isoelectric point electrophoresis). Further, alternatively, an antigen affinity purification method can be used, in which a gel carrier or a membrane to which a protein according to the present invention is chemically bonded is prepared, a material containing an antibody is added thereto, and the adsorbed antibody of interest is eluted and recovered under appropriate conditions.

[0105] Antibody-antigen reaction can be detected according to a known method, for example, as follows. Cells of target lactic acid bacteria are suspended in a solution containing 40 mM Tris-HCl (pH 7.5), 1 mM EDTA, and 150 mM NaCl, and then completely disrupted by vigorously stirring with a sufficient amount of glass beads for several minutes. SDS is added thereto at the final concentration of 0.1% and the suspension is stirred again and then centrifuged to recover the supernatant fraction. The antibody or a Fab′ fragment derived from the antibody is labeled by the linking of a labeling enzyme, such as horseradish peroxidase, a fluorescent substance, biotin, a radioactive isotope or the like, and mixed with the cellular extract of the target lactic acid bacteria to sufficiently bond the antibody to the antigen, after which excessive labeled antibody is removed by washing, and beer-spoilage lactic acid bacteria can be detected by measuring, for example, the activity of the enzyme with which the antibody is labeled. Various methods for such detection, such as an enzyme-linked immunosorbent assay (ELISA) method, a Western blot analysis method, and a radioimmunoassay method, are known and detailed procedures can be found, for example, in Antibodies: A Laboratory Manual by Ed Harlow and David Lane, Cold Spring Harbor Laboratory, 1988.

EXAMPLE

[0106] The present invention is further illustrated by the following examples that are not intended to limit the scope of the invention.

Example 1 Obtaining Beer-Spoilage Lactic Acid Bacteria Gene

[0107] Cells of L. brevis, 3 strains of beer-spoilage lactic acid bacteria and 2 strains of non-beer-spoilage lactic acid bacteria, were cultured statically in 100 ml of MRS medium (Oxoid) to stationary phase and grown cells were recovered, after which the whole DNA was obtained from the cells by the method of Douglas et al. (Applied and Environmental Microbiology, 46, 549-552, 1983). The final DNA concentration was adjusted to about 10 mg/ml to 20 mg/ml and random polymorphic DNA PCR (RAPD PCR) (Nucleic Acid Research, 22, 6531-6535, 1990) was carried out using a 2 ml portion of this DNA as a template. Namely, PCR was performed using 540 kinds of primers from Kit AA to AZ of Operon 10-mer Kits (Operon), i.e., primers for genetic mapping comprising random 10-mer synthetic DNAs. A 20 ml portion of each primer was used at a concentration of 20 mM and a reaction solution was prepared in a total volume of 50 ml. A PCR reaction reagent used was a Takara Ex Taq kit from Takara Shuzo Co., Ltd. and a reaction apparatus used was Gene Amp PCR System 9600 from Perkin Elmer. The reaction program was 45 cycles at 94° C. for 1 minute, at 36° C. for 1 minute, and at 72° C. for 2 minutes. After the reaction was completed, the reaction solution was subjected to electrophoresis with 1% agarose gel, the resulting gel was stained with an ethidium bromide solution, and then amplified bands were analyzed to select primers that recognized a gene specific to beer-spoilage lactic acid bacteria, namely, primers that generated bands having a common size for the template DNAs from the 3 strains of beer-spoilage lactic acid bacteria but did not generate bands having a common size for the template DNAs from the 2 strains of non-beer-spoilage lactic acid bacteria, among the abovementioned 540 primers. Further, PCR was performed in the same way with the primers thus primarily selected, using DNAs extracted from 12 strains of beer-spoilage lactic acid bacteria and 8 strains of non-beer-spoilage lactic acid bacteria as a template DNA to more closely select primers which generate bands specific to beer-spoilage lactic acid bacteria.

[0108] Base sequences of the finally selected primers were as follows: OPAT 07 5′-ACTGCGACCA-3′ (SEQ ID NO: 8) OPAR 12 5′-GGATCGTCGG-3′ (SEQ ID NO: 9) OPAX 05 5′-AGTGCACACC-3′ (SEQ ID NO: 10)

[0109] Next, bands specific to beer-spoilage lactic acid bacteria, which were generated by the abovementioned PCR, were extracted from the agarose gel, recovered using a Mag Extractor PCR and Gel Clean Up kit from Toyobo Co., Ltd, and then cloned into the pCRII vector from Invitrogen Corp.

[0110] Base sequences of the bands specific to beer-spoilage lactic acid bacteria thus cloned were determined, which revealed mutually common sequences. It was confirmed that the regions amplified by three different primers were actually a single region on a chromosome.

[0111] Accordingly, the whole DNA was prepared from beer-spoilage lactic acid bacteria, L. brevis strain L50, using the method of Douglas et al. (Applied and Environmental Microbiology, 46, 549-552, 1983) and this DNA was partially digested with restriction enzyme MboI, after which fragments of 15 to 20 kb were recovered and ligated to the cosmid vector pJB8 to transform E. coli DH1 and thus a genomic library was completed. Screening of this genomic library was carried out using a band amplified with the abovementioned OPAT 07 primer as a probe to obtain a DNA fragment having the restriction map shown in FIG. 1. The base sequence of this fragment was determined (SEQ ID NO: 79). Positions of ORF1 through ORF10 in the nucleotide sequence of SEQ ID NO: 79 are as follows:

[0112] ORF1: from position 4406 to 5353

[0113] ORF2: from position 5363 to 7297

[0114] ORF3: from position 7313 to 7717

[0115] ORF4: from position 8260 to 7766

[0116] ORF5: from position 8778 to 9056

[0117] ORF6: from position 8546 to 8755

[0118] ORF7: from position 9496 to 9867

[0119] ORF8: from position 3939 to 3022

[0120] ORF9: from position 918 to 2204

[0121] ORF10: from position 723 to 205

[0122] Functions of the ORFs found in the above-mentioned base sequences were estimated by comparison with a DNA sequence database such as GENBANK. Results are as follows:

[0123] ORF1: This frame represents 315 amino acids (SEQ ID NO: 3) and presumably encodes a protein having the function of glucosyltransferase or dolichol phosphate mannose synthetase since it shows 69% homology to a protein of estimated molecular mass of 38.5 kDa which is analogous to dolichol mannose synthetase of Bacillus subtilis.

[0124] ORF2: This frame represents 644 amino acids (SEQ ID NO: 5) and shows no homology to any protein in the database used for comparison.

[0125] ORF3: This frame represents 134 amino acids (SEQ ID NO: 7) and presumably encodes a protein having the function of a teichoic acid galactosyltransferase since it shows 63% homology to Listeria monocytogenes gtcA, or teichoic acid glycosylated protein.

[0126] ORF4: This frame represents 176 amino acids and presumably encodes a protein having the function of a nicking enzyme since it has 86% homology to Lactococcus lactis traA.

[0127] ORF5: This frame represents 69 amino acids, which shows 76% homology to a predicted protein ORFOO04 of Lactococcus lactis plasmid pMRC01 (Molecular Micro Biology, 4, 1029-1038, 1998).

[0128] ORF6: This frame represents 92 amino acids, which shows 77% homology to a predicted protein ORFOO03 of Lactococcus lactis plasmid pMRCO1 (Molecular Micro Biology, 4, 1029-1038, 1998).

[0129] ORF7: This frame represents 123 amino acids and presumably encodes a protein having the function of a transposase since it has 62% homology to OrfA of Caulobacter crescentus IS298.

[0130] ORF8: This frame represents 305 amino acids and presumably encodes a protein having the function of a transposase since it shows 62% homology to a predicted transposase of Leuconostoc lactis IS1070.

[0131] ORF9: This frame represents 428 amino acids and shows no homology to any protein in the database used for comparison.

[0132] ORF10: It represents 172 amino acids and presumably encodes a protein having the function of a transcription regulatory factor since it has 49% homology to Lactococcus lactis yxcB.

[0133] Among them, ORF1, ORF2, and ORF3 presumably relate to some sugar chain synthesis since these three ORFs are present within a single operon.

Example 2 Distinction of Beer-Spoilage Lactic Acid Bacteria by PCR

[0134] The following primers were designed from each ORF disclosed in FIG. 1: ORF1-1 5′-GTCAGCGTGCCGACATCCTG-3′ (SEQ ID NO: 11) ORF1-2 5′-TGTATTCACCAATCACCCCG-3′ (SEQ ID NO: 12) ORF2-1 5′-GCCCCGACTTGACCATTTGT-3′ (SEQ ID NO: 13) ORF2-2 5′-TTAGCGGGTGAGCAGCGAGC-3′ (SEQ ID NO: 14) ORF3-1 5′-ACAGCCTTGCGCTACCTGAT-3′ (SEQ ID NO: 15) ORF3-2 5′-TTCACAATCAGCGGCGAACC-3′ (SEQ ID NO: 16) ORF4-1 5′-TGAGTTTTAGTAATATTAGT-3′ (SEQ ID NO: 17) ORF4-2 5′-AGCCAAGCTTGATGCCGGCA-3′ (SEQ ID NO: 18) ORF5-1 5′-AAAGTAACTTAGAAAAACAA-3′ (SEQ ID NO: 19) ORF5-2 5′-ATGATCTACGGACTTTACCT-3′ (SEQ ID NO: 20) ORF6-1 5′-TCAATATGAAAAACTAGTCGAGCAG-3′ (SEQ ID NO: 21) ORF6-2 5′-TTATGGACGTTAACATAGTCAGCA-3′ (SEQ ID NO: 22) ORF7-1 5′-GGAAGATGCTCAGTGGGACCGAATC-3′ (SEQ ID NO: 23) ORF7-2 5′-GCCTTTTGATGCGCTCGAACGAT-3′ (SEQ ID NO: 24) ORF8-1 5′-TCACAGAAAGATTAAGTCGGCAACA-3′ (SEQ ID NO: 25) ORF8-2 5′-TCTAATTCTTTGGCGCTAACCGTC-3′ (SEQ ID NO: 26) ORF9-1 5′-AATTGAAAGTAAGTTGCGAAAGAAA-3′ (SEQ ID NO: 27) ORF9-2 5′-GGCGAACCGTGAACAAATAG-3′ (SEQ ID NO: 28) ORF10-1 5′-TACAATTAGTAAGACAACAGGGATT-3′ (SEQ ID NO: 29) ORF10-2 5′-TCAGGCAATTCTTGTTCATC-3′ (SEQ ID NO: 30)

[0135] Cells of L. brevis, P. damnosus and a single species of lactic acid bacteria, for which an exact taxonomic species name was unknown, as shown in Tables 1, 2 and 3 were cultured and the whole DNAs were obtained by the method of Douglas et al. (Applied and Environmental Microbiology, 46, 549-552, 1983). PCR was carried out with a primer corresponding to each of the abovementioned ORFs using about 0.1 mg of each of these DNAs as a template DNA. A reaction reagent used was a Takara Ex Taq kit from Takara Shuzo Co., Ltd. and a reaction apparatus used was Gene Amp PCR System 9600 from Perkin Elmer. The reaction program was 25 cycles at 94° C. for 30 seconds, at 60° C. for 30 seconds, and at 72° C. for 1 minute. After the reaction was completed, the reaction solution was subjected to electrophoresis with 1.5% agarose gel to examine the presence or absence of a band specific to beer-spoilage lactic acid bacteria. Further, primers were synthesized according to hitA and horA genes, which had been reported as a marker gene for distinguishing beer-spoilage lactic acid bacteria prior to the present invention, and used in a similar experiment to compare results. The results are shown in Tables 1, 2 and 3. TABLE 1 Determination test for beer-spoilage bacteria for Lactobacillus brevis strains Beer- ORF1- ORF2- ORF3- ORF10- spoilage 1,2 1,2 1,2 ORF4-1,2 ORF5-1,2 ORF6-1,2 ORF7-1,2 ORF8-1,2 ORF9-1,2 1,2 hitA-1,2 horA-1,2 Strains ability primer primer primer primer primer primer primer primer primer primer primer primer L42 − − − − − + + + − − − − + L52 − − − − − + + + − − − − + L57 − − − − − + + + − − − − + L62 − − − − − + + + − − − − + L107 − − − − − + + − − − − − + H10 − − − − − − − + − − − − + H14 − − − − − − − + − − − − + L37 + + + + + + + + + + + + + L38 + + + + + + + + + + + + + L40 + + + + + + + + + + + + + L41 + + + + + + + + + + + + + L43 + + + + + + + + + + + + + L45 + + + + + + + + + + + + + L46 + + + + + + + + + + + + + L49 + + + + + + + + + + + + + L49 + + + + + + + + + + + + + L50 + + + + + + + + + + + + + L53 + + + + + + + + + + + + + L58 + + + + + + + + + + + + +

[0136] TABLE 2 Determination test for beer-spoilage bacteria for Pediococcus damnosus strains Beer- ORF1- ORF2- ORF3- ORF10- spoilage 1,2 1,2 1,2 ORF4-1,2 ORF5-1,2 ORF6-1,2 ORF7-1,2 ORF8-1,2 ORF9-1,2 1,2 hitA-1,2 horA-1,2 Strains ability primer primer primer primer primer primer primer primer primer primer primer primer B27 − − − − − + − − − + + + + TB6 − − − − − + − − − + + + + TB23 − − − − − + + − − + − + + TB25 − − − − − + + − − + + + + TB30 − − − − − + + − − + + + + B2 + + + + + + + − + + + + + B3 + + + + + + + − + + + + + B4 + + + + + + + − + + + + + B11 + + + + + + + − + + + + + B13 + + + + + + + − + + + + + B15 + + + + + + + − + + + + + B16 + + + + + + + − + + + + + B20 + + + + + + + − + + + + + B22 + + + + + + + − + + + + + B23 + + + + + + + − + + + + + TB2 + + + + + + + − + + + + + PD1 + + + + + + + − + + + + + PD2 + + + + + + + − + + + + +

[0137] TABLE 3 Determination test for beer-spoilage bacteria for strains of lactic acid bacteria for which species name is unknown Beer- ORF1- ORF2- ORF3- ORF10- spoilage 1,2 1,2 1,2 ORF4-1,2 ORF5-1,2 ORF6-1,2 ORF7-1,2 ORF8-1,2 ORF9-1,2 1,2 hitA-1,2 horA-1,2 Strains ability primer primer primer primer primer primer primer primer primer primer primer primer 4 − − − − − + + − − − − − − 5 − − − − − + + − − − − − − 6 − − − − − + + − − − − − − 7 − + + + + + + − + + + − − 9 − − − − − + + − − − − − − 10 − − − − − + + − − − − − − 12 − − − − − + + − − − − − − 15 − − − − − + + − − − − − − 19 − − − − − + + − − − − − − 8 + + + + + + + − + + + − − 11 + − − − − − + − − − − − − 16 + + + + + + + − + − − − − 21 + + + + + + + − + + + − − 22 + + + + + + + − + + + − − 23 + + + + + + + − + + + − −

[0138] As a result, as shown in Tables, when the operon comprising ORF1, ORF2 and ORF3 was used as a marker, beer-spoilage lactic acid bacteria could be distinguished at a markedly high frequency for both L. brevis and P. damnosus. Further, many beer-spoilage lactic acid bacteria could be distinguished for the strains of taxonomically unknown single species lactic acid bacteria, although the frequency of the distinction was slightly low.

[0139] Next, when ORF4 and ORF8 each neighboring this operon region were used as a marker, beer-spoilage lactic acid bacteria could be distinguished at completely the same frequency as with the genes in the operon.

[0140] On the other hand, when the horA gene was used as a marker, the bands were detected for all of the L. brevis and P. damnosus strains tested, while absolutely no band was detected for the strains of taxonomically unknown single species lactic acid bacteria.

[0141] When the hitA gene was used as a marker, beer-spoilage lactic acid bacteria could be distinguished for L. brevis at completely the same frequency as with ORF1 through ORF4 and ORF8; however, the bands were detected for all strains of P. damnosus while absolutely no band was detected for the strains of taxonomically unknown single species lactic acid bacteria.

[0142] From the results above, it was revealed that the genes of ORF1 through ORF4 and ORF8 disclosed by the present invention were superior to the previously reported genes as a marker for beer-spoilage lactic acid bacteria.

[0143] Further, when ORF9 and ORF10 were used as a marker, beer-spoilage lactic acid bacteria could also be distinguished at a markedly high frequency if limited solely to the strains of L. brevis. When ORF5, ORF6 and ORF7 were used as a marker, no beer-spoilage lactic acid bacteria could be distinguished.

Example 3 Construction of Oligonucleotides for Other Primers and Probes for Regions Highly Specific to Beer-Spoilage Lactic Acid Bacteria

[0144] Primer pairs were designed based on the base sequences of regions of ORF1, ORF2, ORF3, ORF4 and ORF8 having particularly high specificity to beer-spoilage lactic acid bacteria. If necessary, primer pairs can be designed using software for designing primers (for example, OLIGO primer analysis software ver. 6.0, National Biosciences, Inc.) or the like.

[0145] Oligonucleotides were prepared by chemical synthesis and the obtained primer pairs were used for a PCR method. PCR primer pairs for each ORF region and hybridization conditions are as follows. These oligonucleotides can be used singly as a probe.

[0146] (1) ORF1-Related PCR Primers 5′-TTACTGGCCGTTGAAG-3′ (SEQ ID NO: 31) 5′-TGAGCTTGCCGATGT-3′ (SEQ ID NO: 32)

[0147] Conditions for PCR reaction are 25 cycles of 30 seconds at 94° C., 30 seconds at 50° C. and 1 minute at 72° C. 5′-GATGCCGACCTCCAAGATGA-3′ (SEQ ID NO: 33) 5′-CATGCCCACCGCCAGTAG-3′ (SEQ ID NO: 34)

[0148] Conditions for PCR reaction are 25 cycles of 30 seconds of 94° C., 30 seconds at 60° C. and 1 minute at 72° C. 5′-CCGACTTCCGCCTGATG-3′ (SEQ ID NO: 35) 5′-GGTGAGCTTGCCGATGTATT-3′ (SEQ ID NO: 36)

[0149] Conditions for PCR reaction are 25 cycles of 30 seconds at 94° C., 30 seconds at 60° C. and 1 minute at 72° C. 5′-CGCGCAAACCGTCCTC-3′ (SEQ ID NO: 37) 5′-AGCTTGCCGATGTATTCACC-3′ (SEQ ID NO: 38)

[0150] Conditions for PCR reaction are 25 cycles of 30 seconds at 94° C., 30 seconds at 60° C. and 1 minute at 72° C. 5′- TCGCCGGCATGAGTGAAGTCGTGAA-3′ (SEQ ID NO: 39) 5′- CGGCGCAATCGTTAGGCTGGTGAT-3′ (SEQ ID NO: 40)

[0151] Conditions for PCR reaction are 25 cycles of 30 seconds at 94° C., 30 seconds at 65° C. and 1 minute at 72° C.

[0152] (2) ORF2-related PCR primers 5′- GCGCTGTTGGTGGTAG-3′ (SEQ ID NO: 41) 5′- CTGGGCTGCTTGATG-3′ (SEQ ID NO: 42)

[0153] Conditions for PCR reaction are 25 cycles of 30 seconds at 94° C., 30 seconds at 50° C. and 1 minute at 72° C. 5′- TTACTGGCGATGCTGA-3′ (SEQ ID NO: 43) 5′- CTTGGGGATGGTTTTC-3′ (SEQ ID NO: 44)

[0154] Conditions for PCR reaction are 25 cycles of 30 seconds at 94° C., 30 seconds at 50° C. and 1 minute at 72° C. 5′-GTCGCCGTTTGCCATC-3′ (SEQ ID NO: 45) 5′-CGCTTGGGGATGGTTT-3′ (SEQ ID NO: 46)

[0155] Conditions for PCR reaction are 25 cycles of 30 seconds at 94° C., 30 seconds at 55° C. and 1 minute at 72° C. 5′-TCGTGGCCTTCGGTTTCTTT-3′ (SEQ ID NO: 47) 5′-CGCTTGGGGATGGTTTTCA-3′ (SEQ ID NO: 48)

[0156] Conditions for PCR reaction are 25 cycles of 30 seconds at 94° C., 30 seconds at 65° C. and 1 minute at 72° C. 5′-CATCCGGTTGTGGGTAGTGAAGTTA-3′ (SEQ ID NO: 49) 5′-GTGGCAAGGTTAGTGAGGGTGAC-3′ (SEQ ID NO: 50)

[0157] Conditions for PCR reaction are 25 cycles of 30 seconds at 94° C., 30 seconds at 65° C. and 1 minute at 72° C.

[0158] (3) ORF3-related PCR primers 5′-GCCTTGCGCTACCTG-3′ (SEQ ID NO: 51) 5′-GTGTCCGCCAGCAGT-3′ (SEQ ID NO: 52)

[0159] Conditions for PCR reaction are 25 cycles of 30 seconds at 94° C., 30 seconds at 50° C. and 1 minute at 72° C. 5′-TCTTCGGCCTGACTCACCTC-3′ (SEQ ID NO: 53) 5′-GCACGATGACGACGACCTG-3′ (SEQ ID NO: 54)

[0160] Conditions for PCR reaction are 25 cycles of 30 seconds at 94° C., 30 seconds at 60° C. and 1 minute at 72° C. 5′-CTCGCGATGCCGTGGTTCTG-3′ (SEQ ID NO: 55) 5′-CCGTGTCCGCCAGCAGTGA-3′ (SEQ ID NO: 56)

[0161] Conditions for PCR reaction are 25 cycles of 30 seconds at 94° C., 30 seconds at 65° C. and 1 minute at 72° C. 5′-CCTTGCGCTACCTGATTGTTGGAG -3′ (SEQ ID NO: 57) 5′-CATAATTGAGCACGATGACGACGAC-3′ (SEQ ID NO: 58)

[0162] Conditions for PCR reaction are 25 cycles of 30 seconds at 94° C., 30 seconds at 65° C. and 1 minute at 72° C.

[0163] (4) ORF4-related PCR primers 5′-TGAATGGGCGAGTGAT-3′ (SEQ ID NO: 59) 5′-GGCAGCCAAATCGTG-3′ (SEQ ID NO: 60)

[0164] Conditions for PCR reaction are 25 cycles of 30 seconds at 94° C., 30 seconds at 50° C. and 1 minute at 72° C. 5′-GCCAGTGCCGCTTAT-3′ (SEQ ID NO: 61) 5′-TTCTTTCTGTTCGGATTCAC-3′ (SEQ ID NO: 62)

[0165] Conditions for PCR reaction are 25 cycles of 30 seconds at 94° C., 30 seconds at 50° C. and 1 minute at 72° C. 5′-GTGAATCCGAACAGAAAGAA-3′ (SEQ ID NO: 63) 5′-ACAGCCAGCGAATGC-3′ (SEQ ID NO: 64)

[0166] Conditions for PCR reaction are 25 cycles of 30 seconds at 94° C., 30 seconds at 50° C. and 1 minute at 72° C. 5′-GATAAGGAAGGTCGCCACTA-3′ (SEQ ID NO: 65) 5′-GCAGCCAAATCGTGATG-3′ (SEQ ID NO: 66)

[0167] Conditions for PCR reaction are 25 cycles of 30 seconds at 94° C., 30 seconds at 55° C. and 1 minute at 72° C. 5′-AAAGGACGAAGTGCGATTGCCAGTG-3′ (SEQ ID NO: 67) 5′-CGTTCATCACAGCCAGCGAATGC-3′ (SEQ ID NO: 68)

[0168] Conditions for PCR reaction are 25 cycles of 30 seconds at 94° C., 30 seconds at 65° C. and 1 minute at 72° C.

[0169] (5) ORF8-related PCR primers 5′-GCGACGGTCTCTGTT-3′ (SEQ ID NO: 69) 5′-GTTTCTTACCCGATTGC-3′ (SEQ ID NO: 70)

[0170] Conditions for PCR reaction are 25 cycles of 30 seconds at 94° C., 30 seconds at 50° C. and 1 minute at 72° C. 5′-CGACGGTCTCTGTTGAA-3′ (SEQ ID NO: 71) 5′-CCACTAACTTGCCTCACAAT-3′ (SEQ ID NO: 72)

[0171] Conditions for PCR reaction are 25 cycles of 30 seconds at 94° C., 30 seconds at 50° C. and 1 minute at 72° C. 5′-GCTATCGCTGTCTTTTTGAA-3′ (SEQ ID NO: 73) 5′-AATTTTTCGCTCCTTTGGT-3′ (SEQ ID NO: 74)

[0172] Conditions for PCR reaction are 25 cycles of 30 seconds at 94° C., 30 seconds at 60° C. and 1 minute at 72° C. 5′-TGGCAGACGTCAAGTATTTGTTCAC-3′ (SEQ ID NO: 75) 5′-TCAATTTTTCGCTCCTTTGGTATGA-3′ (SEQ ID NO: 76)

[0173] Conditions for PCR reaction are 25 cycles of 30 seconds at 94° C., 30 seconds at 65° C. and 1 minute at 72° C. 5′-GAAATTCATCAAGTCACGCCCTAT-3′ (SEQ ID NO: 77) 5′-TCTCAATTTTTCGCTCCTTTGGTAT-3′ (SEQ ID NO: 78)

[0174] Conditions for PCR reaction are 25 cycles of 30 seconds at 94° C., 30 seconds at 65° C. and 1 minute at 72° C.

1 79 1 8056 DNA Lactobacillus brevis 1 ttagcttagc aaacaaagga ttcctgtaat tgcatgttgt atttgacttg tatcaggcaa 60 ttcttgttca tcaattaggt aattaaaact acccagaata agtgaagtta ataatgacat 120 ttcaaactta tcaggttttt cctccagcat acttacaaga ttgtttctta aaaccctttt 180 tatctcacta ctcaaatctt tgctatccaa ttgaatagag cgcaatgcta aaatcttttc 240 acgttgtttg attaaaagat tttttatatc aggtgccaat atggatgtaa tgctcaaaaa 300 gttttgattt tgtttattta aattacttct tttcttcaat actgaatcat aagtcgacac 360 aaaatcctga atcattttcg cggccaagtg atacttatcc tgatagtgcc tataaaatgt 420 ctgacgattg atcaaggctt tattagagat atcaattacc gatacattat taaatccctg 480 ttgtcttact aattgtataa aagaattttc aattaacatt tctgttctct tatttcttaa 540 atcagtcata agttcctcct tttctccaaa catcagtata gatccatatg taacttaagt 600 atacacaaat aacattttgt atgcctaagt gacgtttcaa aaaaagttgt ctatgtcttt 660 ccaaaataat gtccactatt attagtactt agcttaaaag ggaggaatat cagatgttcg 720 atgtaattcg tagtaaaaga tattggttag cattattgct tgttggagca attattggaa 780 tagtttcatt tgccttcatt ggcatacgta actctgtcaa agtaaaacaa attcctgtag 840 cacttgtcaa cgaagacaaa ggagctctca gcaataaaat tgaaagtaag ttgcgaaaga 900 aattcaatgg aaaagattca aaaatcaaat gggtatctcc acaaaaagat ggttttaatg 960 atcaaaagta ttatggggct ttcattatta gatcaggatt ttcaaaagag ttacagcagc 1020 aaaatgaatc gctaaaggcc caaattatta gccaaaagct cactactctt caaaaaaagg 1080 agaaattacc agattctgca aaatcaaaat tgcttcaagc taaatttaaa tcacaattgc 1140 ttcaagctaa atttgttaca cagaaacccg ttcaccctgc acagattaag atcagtatta 1200 atcaaggaat gaatgctcaa atatcgcaat tgctatccca agcacttcct aagattgcaa 1260 atgcgctttc atcacgaatt agcgcacagc aacaaagtgt tcttagcaaa aataagatta 1320 atttatccgc aaaatcttgg gatttggttt cgacccctat tagtgtatct actcatgagt 1380 ctaataaaat tgaaaagaac acggttaatg gcacagcacc catgcttcta gtggcattgg 1440 cgtggtttag cgctttaatt ccctctctta ttttatggcg cgaacacaca aaaagaagcg 1500 cttcaaaatt tttaaatgct acaacaataa ctagtcaact aattaccggt ttggtagcaa 1560 gtattctttc agcaacagtt gggttcttat ttgttaatgt atgctttaac ctaacaattc 1620 caaacccaat taactttatc ggattaatgt ctattagtat ctttgtcttt tatcttatta 1680 taacgtgtgt cctggattgg ttgggatttg ctttctaccc attactactc gtagtctggc 1740 tcctagcaat ttccgtgata tcttatgcac cggaaaccct tgatcctttg taccgaaagg 1800 gaatttacag ttgggttcca atgcgattta gcatgcaaac actaacaaat actttgtatt 1860 tccataatgg atcgagtacc accatgtcat cattattagt cttgttaata attggatttg 1920 tcgctgctat cttgatgtat agttcaggat acttaaaaca ctatttgttc acggttcgcc 1980 cacaccgcaa aattaaataa ttaaacaaaa catgaaaccc aattattcat atcattaaaa 2040 atagccaatc aattgattga ctattttttg atgatccatt tagcaacaat tccctcaaaa 2100 aaaaagagag tgtgaaatat tttgtgtaaa tgtatataaa aattctgaat tgtatagagg 2160 cagagaatgt tcttgaggta taccctattg gctaatcaca cataaaagct ttcttatatc 2220 attatttttt tggcaggtac tgtgtaggtg gaaatgaacg aatgctttaa tagttttgtg 2280 tcacaaaata aattttggag attttcagca ctccagtagt tcaatgattc ctggcatatt 2340 ccatcagcaa gtttatatat tgcgtctttt aacaattgga gcttactttc agaaactaca 2400 tgaactttaa agttggtatg tgcatctgaa gcaaagatga tagaggggtg gaaaataatg 2460 gaatagaaag tttgactctg atatttttcc gcaaaccaat tcgaagatgt agttatttgt 2520 tctacatcac ctttaaagat ttcatcacta gctcgtcgat ttttatcttc tatgacaatc 2580 tgtgtgcttt gatccagcca aagattatca ggaccgcctt ctttgatagc cgtttctgcc 2640 tcaggctgac tagaatcaaa tcctaataat tctcctaatc tttgaatgct tgctctaaag 2700 tttgtttcgt cggcatagtt tgtataaagc aggtcatcat taatagctcg aatatgtgta 2760 gctaaatcat tgctatcttc gaagtcatac ttttttatat atggtagatt gcaagaatta 2820 accgaacact gcgaactgct gaaaaacctt tcgtggtgat tgccaattga gtgttttcat 2880 tggtcttgca ttaatccaat gattaatttg atctaattct ttggcgctaa ccgtctcaat 2940 ttttcgctcc tttggtatga atcgccgaac gtatcggttt aatatctcat tactcccacg 3000 ttcttctggt gagtatgggt gtgcaaagta aactggaatg cctagttttt gctcgatttc 3060 atcatatttt acaaactctc gtcctcgatc aacagtgatt gacttagcat tctcgatccc 3120 ttgaaaaaaa tgaattaata ctggtgtcac tgcctgacta ttgcgaccac taacttgcct 3180 cacaatatgt tgccgactta atctttctgt gattgtgaca agcacttcac cacgtttctt 3240 acccgattgc atcgtatcaa cttcaaagtg accaaaatcc tgccgtgctt gaacactttt 3300 gggtcttgat tcaattgaac ggccgtgaac aaatacttga cgtctgccat cagactgacg 3360 tttttgacga atacctttgt caggtaaatc agctaatgac agtttgagtc ggccagcatt 3420 gagccaatta taaatcgttt tgaaaggtaa ccctaaaaca tgagcagcag tttctggtga 3480 ccactttaaa atgccaatgt gctcattcaa aaagacagcg atagctggtg ttagtgtgtg 3540 atgacggcca cgtagatgac gttttttcaa tgccaatgca tgagcaatat cagctttata 3600 gggcgtgact tgatgaattt caacagagac cgtcgcaggt gatcggttaa taaaacgcgc 3660 gatggcacga atcgaataat tcaattccag caaagtttga atgacagtac gttcttgaga 3720 tgataaacta gtcatgagtc gcagttcctt tatggttgtt ttggacaatt accattaaag 3780 gcacaattca catagggaga agaccattga aatccaccat tttactagtc gaagacgagg 3840 cgggcttagc cgactcactc aaaaccgaat ttgagctcga gaacttcaat gtgttctggg 3900 ccaatgacgg cctaatcgcg ctggacatgt tccggcaaaa cgaggcccag attgatttaa 3960 tcattttgga ctggatgctg ccgcacattc aagatcaaga aacccagctg attactgtat 4020 tgattggcat tggctggatc gcgatggtgg cgatatcgcg ggtgtatctg cgcgaccact 4080 acctctctga tgtgctcgcc agtgtctgct tagctagccg ctggtggttg ctggtcacac 4140 ctgcggaagc ctttattcaa gctaaaatgc ggcagttttt accggaaggg atgttgaaat 4200 catggccaca tcaaaattaa cgattgttgt tcctgcttac aatgaagaag aggtgctgac 4260 gtcctcggtg caaaaattac tggccgttga agaccaaatc gccgcgcaaa ccgtcctcgg 4320 tcagcgtgcc gacatcctga tcgtcgatga tggttccatg gatcacacct gggacatcat 4380 cgaaaaactg cacgccatga attctcgcgt gcgcggactg cgtttttccc gcaacttcgg 4440 ccaccagtcg gcgctgatcg ccggcatgag tgaagtcgtg aaaaccgccg atatgattgt 4500 caccatcgat gccgacctcc aagatgatcc cgacaaaatc ggcgacatgg tggatgccta 4560 tgcggatggc gccgacatcg tctacggcgt ccggaacaac cgggaaaccg acagctggtt 4620 caagcgcacc acggcccaag gctactacaa gacactcaag ctgctgggcg tcgaactcgt 4680 gcccaatcac gccgacttcc gcctgatgtc caagcgcgcc gttgaaacct tcctgcagta 4740 tccagaacgc aacattttca ttcgcggcct gattcctaag ctcggcttca aaactgccga 4800 agtcttctac aagcgcacac cgcgcatggc cggcgaatcc aagtacccgc tgaaaaagat 4860 gctggctttt gcctgggacg gcatcaccag cctaacgatt gcgccggtgc ggctcattct 4920 cattctgggt accttgtctt gcctactggc ggtgggcatg gtggtttacg ccattgtcat 4980 gaaaatgctg gggctcaccg tgcacggctg gtcgttgttg atggtgtcgc tgtggttcgt 5040 tggcggcatc caaatgatca gcctcggggt gattggtgaa tacatcggca agctcaccac 5100 cgaagttaaa catcgcccgc gctacacggt gcaaacgatt ctggattgag gtgagggtat 5160 gaaaaagatt aaacccgcct ttctgccagc aattttaatc ctctgcctgc tcattggcag 5220 catcggtaac ctgaccagtg tgctcggggt gccggcgctg ttggtggtag tgctcctggg 5280 cgcggggctt tacttcgggg cgccccgact tgaccatttg tctacaaggc agctgcgctg 5340 gggcattggc cttggcttac tggcgatgct gattgcccag gtggtcgtgt tgcacgtgat 5400 gcccaacacc gtttaccacg atccgtaccg ggtactgtcg caagccgacc agctcgccgc 5460 cggccacatg acctgggata tcacctactt ctggcgctac gccaataacg tgccgctggc 5520 ttatctgctc tccctgtggt tgcggctgac gcaactggtg ggcttaagca ccaatctttc 5580 ggtgcacctg ctgagtatct tggtgttgga cagctttatt gccctggcgc tgcatacgat 5640 ttggcagctc agccagcgcg ccagcctgct ggtcgtggcc ttcggtttct ttgccttgtc 5700 gccgtttgcc tacacctact acctgcaagt cttttactcc gacttaccga cgatgctggt 5760 gctgctcatc atcatacgca gcctgctgaa ctggtcgcag aaaacatcgc gccagcgctg 5820 gtttgccggc agcggactag ttgttgccgt gatgctcggc gccatgctca agcctaatct 5880 ggtggtcttg ttgccagctc tgctgattgt cggcctgatt ctggcccgtc agcacctctg 5940 gcgacaagcc aaactgaccc tgcccatcct cttgattgtg ctgggcttcg ggctgagtct 6000 gccggcgacc aaagtctttg acgtggcagc caattatcaa ccccgcaccg ccttttcgtt 6060 cccggcgacc cactggatct tgatgggcta caaccagcac agcaacggcg gctactccgg 6120 caaggatgtc ggacgtgcca tcaagcagcc cagccaagcc gaccgccagc ggtacaattt 6180 gaaaaccatc cccaagcgca tcaaaactct cggggtggtt ggcgtcatcc ggttgtgggt 6240 agtgaagtta ggcatcgtgc tcaatgtcca aggcattcag cgctggtaca acggcggctt 6300 ccgcgccgcg cctagttggt acagtaatca tgctggcttc tatcagggac tgaccgtgat 6360 tggctatgtg gccgcgaccc tgctcatgtg gggcgcactg atgctgaagc tcttgcggtg 6420 gcggccagat ctgaccgacc cgcatcaaat ccttgcactg ctggcggtga ccactgccct 6480 tggctacctg gctttccaca ccctactgtg ggaagttgaa ccgcgctatg gtcaagccat 6540 tttgccgctg ctctgggtgg ctttggcggc catcccgcgt caggccagcc agtcgcgtcc 6600 ccgctgggcg aaccaagcta gcctcctcaa tggcgccact gcttcactcg tcgcctttgg 6660 ggccgctggt gtgcttggcg ctcagctgcc acaaaagcaa gtgattgccg cccagcgcag 6720 tcagctatcc gtgcagtatc acgccaagcc caagaccgtg acgccaggca ccgtgctggc 6780 agaggtggtc gatgtgaacg cgccagcgaa ctatttttcc gttcagattc atgctggtag 6840 tcaggtgcaa gtcaccctca ctaaccttgc caccgggcaa cattatcggt taacgatggc 6900 tggcagtgtg gcccgcctgc accaccagct cgccgctggg caatatcgga ttaccgttca 6960 aaacctcacc acccgcggcc agcaggtcga tgtgacccac acctaccatt atcagctcgc 7020 tgctcacccg ctaacggtga acggccaatc gcagcccacc gcctcgttga tttatacctg 7080 catgcagcgc tgagaaagga gcctttttat ggaaaaacca atcactaccc tttacagcaa 7140 atacgataca gccttgcgct acctgattgt tggaggcctc accaccggca ttaatgtggt 7200 gctgttcttc ggcctgactc acctcgcgat gccgtggttc tgggcgaaca ttatcgcctg 7260 ggtcctcagc gtgctgtttg ccttcattgc caacaagaaa gtcgtgttca actccgccga 7320 catgaccttc cggactgtgg tcaaagaagg cgccagcttc ttcaccttgc gcggcgcgtc 7380 actgctggcg gacacggcga ttttgttcat cggcctcacc ttaatgcacg gttcgccgct 7440 gattgtgaag ctgatcgacc aggtcgtcgt catcgtgctc aattatggct tcagcaaact 7500 aattttcgct taacgtaaaa atggtcccag cagtggaaac tgccgagacc attttgcttg 7560 gctagccaag cttgatgccg gcatccgcca gcgctgcgtc gaccacgttc atcacagcca 7620 gcgaatgcgc catccgctgc ttggcggcag ccaaatcgtg atgcgcaatc atcgtttcga 7680 acgcgacgaa ctcttcgtac atgcggtgcc ggtcagctac cataccttga tcgacaaaat 7740 tttcttgcac atattttgtc agtaattctt tctgttcgga ttcacttaat tctaccggta 7800 aagccacgtt aaactctttt gcataccgtg agtttgattt acgatctttc ttttcaactt 7860 cattccacaa ctgctctcga tcactcgccc attcaggtga attttttggc gtcaaaataa 7920 agctttctgg catgatcgat cgggcataaa aatagtggcg accttcctta tcatcaaata 7980 gcttttcacc acttcgataa gcggcactgg caatcgcact tcgtccttta ccagcactaa 8040 tattactaaa actcat 8056 2 948 DNA Lactobacillus brevis CDS (1)..(945) 2 atg gcc aca tca aaa tta acg att gtt gtt cct gct tac aat gaa gaa 48 Met Ala Thr Ser Lys Leu Thr Ile Val Val Pro Ala Tyr Asn Glu Glu 1 5 10 15 gag gtg ctg acg tcc tcg gtg caa aaa tta ctg gcc gtt gaa gac caa 96 Glu Val Leu Thr Ser Ser Val Gln Lys Leu Leu Ala Val Glu Asp Gln 20 25 30 atc gcc gcg caa acc gtc ctc ggt cag cgt gcc gac atc ctg atc gtc 144 Ile Ala Ala Gln Thr Val Leu Gly Gln Arg Ala Asp Ile Leu Ile Val 35 40 45 gat gat ggt tcc atg gat cac acc tgg gac atc atc gaa aaa ctg cac 192 Asp Asp Gly Ser Met Asp His Thr Trp Asp Ile Ile Glu Lys Leu His 50 55 60 gcc atg aat tct cgc gtg cgc gga ctg cgt ttt tcc cgc aac ttc ggc 240 Ala Met Asn Ser Arg Val Arg Gly Leu Arg Phe Ser Arg Asn Phe Gly 65 70 75 80 cac cag tcg gcg ctg atc gcc ggc atg agt gaa gtc gtg aaa acc gcc 288 His Gln Ser Ala Leu Ile Ala Gly Met Ser Glu Val Val Lys Thr Ala 85 90 95 gat atg att gtc acc atc gat gcc gac ctc caa gat gat ccc gac aaa 336 Asp Met Ile Val Thr Ile Asp Ala Asp Leu Gln Asp Asp Pro Asp Lys 100 105 110 atc ggc gac atg gtg gat gcc tat gcg gat ggc gcc gac atc gtc tac 384 Ile Gly Asp Met Val Asp Ala Tyr Ala Asp Gly Ala Asp Ile Val Tyr 115 120 125 ggc gtc cgg aac aac cgg gaa acc gac agc tgg ttc aag cgc acc acg 432 Gly Val Arg Asn Asn Arg Glu Thr Asp Ser Trp Phe Lys Arg Thr Thr 130 135 140 gcc caa ggc tac tac aag aca ctc aag ctg ctg ggc gtc gaa ctc gtg 480 Ala Gln Gly Tyr Tyr Lys Thr Leu Lys Leu Leu Gly Val Glu Leu Val 145 150 155 160 ccc aat cac gcc gac ttc cgc ctg atg tcc aag cgc gcc gtt gaa acc 528 Pro Asn His Ala Asp Phe Arg Leu Met Ser Lys Arg Ala Val Glu Thr 165 170 175 ttc ctg cag tat cca gaa cgc aac att ttc att cgc ggc ctg att cct 576 Phe Leu Gln Tyr Pro Glu Arg Asn Ile Phe Ile Arg Gly Leu Ile Pro 180 185 190 aag ctc ggc ttc aaa act gcc gaa gtc ttc tac aag cgc aca ccg cgc 624 Lys Leu Gly Phe Lys Thr Ala Glu Val Phe Tyr Lys Arg Thr Pro Arg 195 200 205 atg gcc ggc gaa tcc aag tac ccg ctg aaa aag atg ctg gct ttt gcc 672 Met Ala Gly Glu Ser Lys Tyr Pro Leu Lys Lys Met Leu Ala Phe Ala 210 215 220 tgg gac ggc atc acc agc cta acg att gcg ccg gtg cgg ctc att ctc 720 Trp Asp Gly Ile Thr Ser Leu Thr Ile Ala Pro Val Arg Leu Ile Leu 225 230 235 240 att ctg ggt acc ttg tct tgc cta ctg gcg gtg ggc atg gtg gtt tac 768 Ile Leu Gly Thr Leu Ser Cys Leu Leu Ala Val Gly Met Val Val Tyr 245 250 255 gcc att gtc atg aaa atg ctg ggg ctc acc gtg cac ggc tgg tcg ttg 816 Ala Ile Val Met Lys Met Leu Gly Leu Thr Val His Gly Trp Ser Leu 260 265 270 ttg atg gtg tcg ctg tgg ttc gtt ggc ggc atc caa atg atc agc ctc 864 Leu Met Val Ser Leu Trp Phe Val Gly Gly Ile Gln Met Ile Ser Leu 275 280 285 ggg gtg att ggt gaa tac atc ggc aag ctc acc acc gaa gtt aaa cat 912 Gly Val Ile Gly Glu Tyr Ile Gly Lys Leu Thr Thr Glu Val Lys His 290 295 300 cgc ccg cgc tac acg gtg caa acg att ctg gat tga 948 Arg Pro Arg Tyr Thr Val Gln Thr Ile Leu Asp 305 310 315 3 315 PRT Lactobacillus brevis 3 Met Ala Thr Ser Lys Leu Thr Ile Val Val Pro Ala Tyr Asn Glu Glu 1 5 10 15 Glu Val Leu Thr Ser Ser Val Gln Lys Leu Leu Ala Val Glu Asp Gln 20 25 30 Ile Ala Ala Gln Thr Val Leu Gly Gln Arg Ala Asp Ile Leu Ile Val 35 40 45 Asp Asp Gly Ser Met Asp His Thr Trp Asp Ile Ile Glu Lys Leu His 50 55 60 Ala Met Asn Ser Arg Val Arg Gly Leu Arg Phe Ser Arg Asn Phe Gly 65 70 75 80 His Gln Ser Ala Leu Ile Ala Gly Met Ser Glu Val Val Lys Thr Ala 85 90 95 Asp Met Ile Val Thr Ile Asp Ala Asp Leu Gln Asp Asp Pro Asp Lys 100 105 110 Ile Gly Asp Met Val Asp Ala Tyr Ala Asp Gly Ala Asp Ile Val Tyr 115 120 125 Gly Val Arg Asn Asn Arg Glu Thr Asp Ser Trp Phe Lys Arg Thr Thr 130 135 140 Ala Gln Gly Tyr Tyr Lys Thr Leu Lys Leu Leu Gly Val Glu Leu Val 145 150 155 160 Pro Asn His Ala Asp Phe Arg Leu Met Ser Lys Arg Ala Val Glu Thr 165 170 175 Phe Leu Gln Tyr Pro Glu Arg Asn Ile Phe Ile Arg Gly Leu Ile Pro 180 185 190 Lys Leu Gly Phe Lys Thr Ala Glu Val Phe Tyr Lys Arg Thr Pro Arg 195 200 205 Met Ala Gly Glu Ser Lys Tyr Pro Leu Lys Lys Met Leu Ala Phe Ala 210 215 220 Trp Asp Gly Ile Thr Ser Leu Thr Ile Ala Pro Val Arg Leu Ile Leu 225 230 235 240 Ile Leu Gly Thr Leu Ser Cys Leu Leu Ala Val Gly Met Val Val Tyr 245 250 255 Ala Ile Val Met Lys Met Leu Gly Leu Thr Val His Gly Trp Ser Leu 260 265 270 Leu Met Val Ser Leu Trp Phe Val Gly Gly Ile Gln Met Ile Ser Leu 275 280 285 Gly Val Ile Gly Glu Tyr Ile Gly Lys Leu Thr Thr Glu Val Lys His 290 295 300 Arg Pro Arg Tyr Thr Val Gln Thr Ile Leu Asp 305 310 315 4 1935 DNA Lactobacillus brevis CDS (1)..(1932) 4 atg aaa aag att aaa ccc gcc ttt ctg cca gca att tta atc ctc tgc 48 Met Lys Lys Ile Lys Pro Ala Phe Leu Pro Ala Ile Leu Ile Leu Cys 1 5 10 15 ctg ctc att ggc agc atc ggt aac ctg acc agt gtg ctc ggg gtg ccg 96 Leu Leu Ile Gly Ser Ile Gly Asn Leu Thr Ser Val Leu Gly Val Pro 20 25 30 gcg ctg ttg gtg gta gtg ctc ctg ggc gcg ggg ctt tac ttc ggg gcg 144 Ala Leu Leu Val Val Val Leu Leu Gly Ala Gly Leu Tyr Phe Gly Ala 35 40 45 ccc cga ctt gac cat ttg tct aca agg cag ctg cgc tgg ggc att ggc 192 Pro Arg Leu Asp His Leu Ser Thr Arg Gln Leu Arg Trp Gly Ile Gly 50 55 60 ctt ggc tta ctg gcg atg ctg att gcc cag gtg gtc gtg ttg cac gtg 240 Leu Gly Leu Leu Ala Met Leu Ile Ala Gln Val Val Val Leu His Val 65 70 75 80 atg ccc aac acc gtt tac cac gat ccg tac cgg gta ctg tcg caa gcc 288 Met Pro Asn Thr Val Tyr His Asp Pro Tyr Arg Val Leu Ser Gln Ala 85 90 95 gac cag ctc gcc gcc ggc cac atg acc tgg gat atc acc tac ttc tgg 336 Asp Gln Leu Ala Ala Gly His Met Thr Trp Asp Ile Thr Tyr Phe Trp 100 105 110 cgc tac gcc aat aac gtg ccg ctg gct tat ctg ctc tcc ctg tgg ttg 384 Arg Tyr Ala Asn Asn Val Pro Leu Ala Tyr Leu Leu Ser Leu Trp Leu 115 120 125 cgg ctg acg caa ctg gtg ggc tta agc acc aat ctt tcg gtg cac ctg 432 Arg Leu Thr Gln Leu Val Gly Leu Ser Thr Asn Leu Ser Val His Leu 130 135 140 ctg agt atc ttg gtg ttg gac agc ttt att gcc ctg gcg ctg cat acg 480 Leu Ser Ile Leu Val Leu Asp Ser Phe Ile Ala Leu Ala Leu His Thr 145 150 155 160 att tgg cag ctc agc cag cgc gcc agc ctg ctg gtc gtg gcc ttc ggt 528 Ile Trp Gln Leu Ser Gln Arg Ala Ser Leu Leu Val Val Ala Phe Gly 165 170 175 ttc ttt gcc ttg tcg ccg ttt gcc tac acc tac tac ctg caa gtc ttt 576 Phe Phe Ala Leu Ser Pro Phe Ala Tyr Thr Tyr Tyr Leu Gln Val Phe 180 185 190 tac tcc gac tta ccg acg atg ctg gtg ctg ctc atc atc ata cgc agc 624 Tyr Ser Asp Leu Pro Thr Met Leu Val Leu Leu Ile Ile Ile Arg Ser 195 200 205 ctg ctg aac tgg tcg cag aaa aca tcg cgc cag cgc tgg ttt gcc ggc 672 Leu Leu Asn Trp Ser Gln Lys Thr Ser Arg Gln Arg Trp Phe Ala Gly 210 215 220 agc gga cta gtt gtt gcc gtg atg ctc ggc gcc atg ctc aag cct aat 720 Ser Gly Leu Val Val Ala Val Met Leu Gly Ala Met Leu Lys Pro Asn 225 230 235 240 ctg gtg gtc ttg ttg cca gct ctg ctg att gtc ggc ctg att ctg gcc 768 Leu Val Val Leu Leu Pro Ala Leu Leu Ile Val Gly Leu Ile Leu Ala 245 250 255 cgt cag cac ctc tgg cga caa gcc aaa ctg acc ctg ccc atc ctc ttg 816 Arg Gln His Leu Trp Arg Gln Ala Lys Leu Thr Leu Pro Ile Leu Leu 260 265 270 att gtg ctg ggc ttc ggg ctg agt ctg ccg gcg acc aaa gtc ttt gac 864 Ile Val Leu Gly Phe Gly Leu Ser Leu Pro Ala Thr Lys Val Phe Asp 275 280 285 gtg gca gcc aat tat caa ccc cgc acc gcc ttt tcg ttc ccg gcg acc 912 Val Ala Ala Asn Tyr Gln Pro Arg Thr Ala Phe Ser Phe Pro Ala Thr 290 295 300 cac tgg atc ttg atg ggc tac aac cag cac agc aac ggc ggc tac tcc 960 His Trp Ile Leu Met Gly Tyr Asn Gln His Ser Asn Gly Gly Tyr Ser 305 310 315 320 ggc aag gat gtc gga cgt gcc atc aag cag ccc agc caa gcc gac cgc 1008 Gly Lys Asp Val Gly Arg Ala Ile Lys Gln Pro Ser Gln Ala Asp Arg 325 330 335 cag cgg tac aat ttg aaa acc atc ccc aag cgc atc aaa act ctc ggg 1056 Gln Arg Tyr Asn Leu Lys Thr Ile Pro Lys Arg Ile Lys Thr Leu Gly 340 345 350 gtg gtt ggc gtc atc cgg ttg tgg gta gtg aag tta ggc atc gtg ctc 1104 Val Val Gly Val Ile Arg Leu Trp Val Val Lys Leu Gly Ile Val Leu 355 360 365 aat gtc caa ggc att cag cgc tgg tac aac ggc ggc ttc cgc gcc gcg 1152 Asn Val Gln Gly Ile Gln Arg Trp Tyr Asn Gly Gly Phe Arg Ala Ala 370 375 380 cct agt tgg tac agt aat cat gct ggc ttc tat cag gga ctg acc gtg 1200 Pro Ser Trp Tyr Ser Asn His Ala Gly Phe Tyr Gln Gly Leu Thr Val 385 390 395 400 att ggc tat gtg gcc gcg acc ctg ctc atg tgg ggc gca ctg atg ctg 1248 Ile Gly Tyr Val Ala Ala Thr Leu Leu Met Trp Gly Ala Leu Met Leu 405 410 415 aag ctc ttg cgg tgg cgg cca gat ctg acc gac ccg cat caa atc ctt 1296 Lys Leu Leu Arg Trp Arg Pro Asp Leu Thr Asp Pro His Gln Ile Leu 420 425 430 gca ctg ctg gcg gtg acc act gcc ctt ggc tac ctg gct ttc cac acc 1344 Ala Leu Leu Ala Val Thr Thr Ala Leu Gly Tyr Leu Ala Phe His Thr 435 440 445 cta ctg tgg gaa gtt gaa ccg cgc tat ggt caa gcc att ttg ccg ctg 1392 Leu Leu Trp Glu Val Glu Pro Arg Tyr Gly Gln Ala Ile Leu Pro Leu 450 455 460 ctc tgg gtg gct ttg gcg gcc atc ccg cgt cag gcc agc cag tcg cgt 1440 Leu Trp Val Ala Leu Ala Ala Ile Pro Arg Gln Ala Ser Gln Ser Arg 465 470 475 480 ccc cgc tgg gcg aac caa gct agc ctc ctc aat ggc gcc act gct tca 1488 Pro Arg Trp Ala Asn Gln Ala Ser Leu Leu Asn Gly Ala Thr Ala Ser 485 490 495 ctc gtc gcc ttt ggg gcc gct ggt gtg ctt ggc gct cag ctg cca caa 1536 Leu Val Ala Phe Gly Ala Ala Gly Val Leu Gly Ala Gln Leu Pro Gln 500 505 510 aag caa gtg att gcc gcc cag cgc agt cag cta tcc gtg cag tat cac 1584 Lys Gln Val Ile Ala Ala Gln Arg Ser Gln Leu Ser Val Gln Tyr His 515 520 525 gcc aag ccc aag acc gtg acg cca ggc acc gtg ctg gca gag gtg gtc 1632 Ala Lys Pro Lys Thr Val Thr Pro Gly Thr Val Leu Ala Glu Val Val 530 535 540 gat gtg aac gcg cca gcg aac tat ttt tcc gtt cag att cat gct ggt 1680 Asp Val Asn Ala Pro Ala Asn Tyr Phe Ser Val Gln Ile His Ala Gly 545 550 555 560 agt cag gtg caa gtc acc ctc act aac ctt gcc acc ggg caa cat tat 1728 Ser Gln Val Gln Val Thr Leu Thr Asn Leu Ala Thr Gly Gln His Tyr 565 570 575 cgg tta acg atg gct ggc agt gtg gcc cgc ctg cac cac cag ctc gcc 1776 Arg Leu Thr Met Ala Gly Ser Val Ala Arg Leu His His Gln Leu Ala 580 585 590 gct ggg caa tat cgg att acc gtt caa aac ctc acc acc cgc ggc cag 1824 Ala Gly Gln Tyr Arg Ile Thr Val Gln Asn Leu Thr Thr Arg Gly Gln 595 600 605 cag gtc gat gtg acc cac acc tac cat tat cag ctc gct gct cac ccg 1872 Gln Val Asp Val Thr His Thr Tyr His Tyr Gln Leu Ala Ala His Pro 610 615 620 cta acg gtg aac ggc caa tcg cag ccc acc gcc tcg ttg att tat acc 1920 Leu Thr Val Asn Gly Gln Ser Gln Pro Thr Ala Ser Leu Ile Tyr Thr 625 630 635 640 tgc atg cag cgc tga 1935 Cys Met Gln Arg 5 644 PRT Lactobacillus brevis 5 Met Lys Lys Ile Lys Pro Ala Phe Leu Pro Ala Ile Leu Ile Leu Cys 1 5 10 15 Leu Leu Ile Gly Ser Ile Gly Asn Leu Thr Ser Val Leu Gly Val Pro 20 25 30 Ala Leu Leu Val Val Val Leu Leu Gly Ala Gly Leu Tyr Phe Gly Ala 35 40 45 Pro Arg Leu Asp His Leu Ser Thr Arg Gln Leu Arg Trp Gly Ile Gly 50 55 60 Leu Gly Leu Leu Ala Met Leu Ile Ala Gln Val Val Val Leu His Val 65 70 75 80 Met Pro Asn Thr Val Tyr His Asp Pro Tyr Arg Val Leu Ser Gln Ala 85 90 95 Asp Gln Leu Ala Ala Gly His Met Thr Trp Asp Ile Thr Tyr Phe Trp 100 105 110 Arg Tyr Ala Asn Asn Val Pro Leu Ala Tyr Leu Leu Ser Leu Trp Leu 115 120 125 Arg Leu Thr Gln Leu Val Gly Leu Ser Thr Asn Leu Ser Val His Leu 130 135 140 Leu Ser Ile Leu Val Leu Asp Ser Phe Ile Ala Leu Ala Leu His Thr 145 150 155 160 Ile Trp Gln Leu Ser Gln Arg Ala Ser Leu Leu Val Val Ala Phe Gly 165 170 175 Phe Phe Ala Leu Ser Pro Phe Ala Tyr Thr Tyr Tyr Leu Gln Val Phe 180 185 190 Tyr Ser Asp Leu Pro Thr Met Leu Val Leu Leu Ile Ile Ile Arg Ser 195 200 205 Leu Leu Asn Trp Ser Gln Lys Thr Ser Arg Gln Arg Trp Phe Ala Gly 210 215 220 Ser Gly Leu Val Val Ala Val Met Leu Gly Ala Met Leu Lys Pro Asn 225 230 235 240 Leu Val Val Leu Leu Pro Ala Leu Leu Ile Val Gly Leu Ile Leu Ala 245 250 255 Arg Gln His Leu Trp Arg Gln Ala Lys Leu Thr Leu Pro Ile Leu Leu 260 265 270 Ile Val Leu Gly Phe Gly Leu Ser Leu Pro Ala Thr Lys Val Phe Asp 275 280 285 Val Ala Ala Asn Tyr Gln Pro Arg Thr Ala Phe Ser Phe Pro Ala Thr 290 295 300 His Trp Ile Leu Met Gly Tyr Asn Gln His Ser Asn Gly Gly Tyr Ser 305 310 315 320 Gly Lys Asp Val Gly Arg Ala Ile Lys Gln Pro Ser Gln Ala Asp Arg 325 330 335 Gln Arg Tyr Asn Leu Lys Thr Ile Pro Lys Arg Ile Lys Thr Leu Gly 340 345 350 Val Val Gly Val Ile Arg Leu Trp Val Val Lys Leu Gly Ile Val Leu 355 360 365 Asn Val Gln Gly Ile Gln Arg Trp Tyr Asn Gly Gly Phe Arg Ala Ala 370 375 380 Pro Ser Trp Tyr Ser Asn His Ala Gly Phe Tyr Gln Gly Leu Thr Val 385 390 395 400 Ile Gly Tyr Val Ala Ala Thr Leu Leu Met Trp Gly Ala Leu Met Leu 405 410 415 Lys Leu Leu Arg Trp Arg Pro Asp Leu Thr Asp Pro His Gln Ile Leu 420 425 430 Ala Leu Leu Ala Val Thr Thr Ala Leu Gly Tyr Leu Ala Phe His Thr 435 440 445 Leu Leu Trp Glu Val Glu Pro Arg Tyr Gly Gln Ala Ile Leu Pro Leu 450 455 460 Leu Trp Val Ala Leu Ala Ala Ile Pro Arg Gln Ala Ser Gln Ser Arg 465 470 475 480 Pro Arg Trp Ala Asn Gln Ala Ser Leu Leu Asn Gly Ala Thr Ala Ser 485 490 495 Leu Val Ala Phe Gly Ala Ala Gly Val Leu Gly Ala Gln Leu Pro Gln 500 505 510 Lys Gln Val Ile Ala Ala Gln Arg Ser Gln Leu Ser Val Gln Tyr His 515 520 525 Ala Lys Pro Lys Thr Val Thr Pro Gly Thr Val Leu Ala Glu Val Val 530 535 540 Asp Val Asn Ala Pro Ala Asn Tyr Phe Ser Val Gln Ile His Ala Gly 545 550 555 560 Ser Gln Val Gln Val Thr Leu Thr Asn Leu Ala Thr Gly Gln His Tyr 565 570 575 Arg Leu Thr Met Ala Gly Ser Val Ala Arg Leu His His Gln Leu Ala 580 585 590 Ala Gly Gln Tyr Arg Ile Thr Val Gln Asn Leu Thr Thr Arg Gly Gln 595 600 605 Gln Val Asp Val Thr His Thr Tyr His Tyr Gln Leu Ala Ala His Pro 610 615 620 Leu Thr Val Asn Gly Gln Ser Gln Pro Thr Ala Ser Leu Ile Tyr Thr 625 630 635 640 Cys Met Gln Arg 6 405 DNA Lactobacillus brevis CDS (1)..(402) 6 atg gaa aaa cca atc act acc ctt tac agc aaa tac gat aca gcc ttg 48 Met Glu Lys Pro Ile Thr Thr Leu Tyr Ser Lys Tyr Asp Thr Ala Leu 1 5 10 15 cgc tac ctg att gtt gga ggc ctc acc acc ggc att aat gtg gtg ctg 96 Arg Tyr Leu Ile Val Gly Gly Leu Thr Thr Gly Ile Asn Val Val Leu 20 25 30 ttc ttc ggc ctg act cac ctc gcg atg ccg tgg ttc tgg gcg aac att 144 Phe Phe Gly Leu Thr His Leu Ala Met Pro Trp Phe Trp Ala Asn Ile 35 40 45 atc gcc tgg gtc ctc agc gtg ctg ttt gcc ttc att gcc aac aag aaa 192 Ile Ala Trp Val Leu Ser Val Leu Phe Ala Phe Ile Ala Asn Lys Lys 50 55 60 gtc gtg ttc aac tcc gcc gac atg acc ttc cgg act gtg gtc aaa gaa 240 Val Val Phe Asn Ser Ala Asp Met Thr Phe Arg Thr Val Val Lys Glu 65 70 75 80 ggc gcc agc ttc ttc acc ttg cgc ggc gcg tca ctg ctg gcg gac acg 288 Gly Ala Ser Phe Phe Thr Leu Arg Gly Ala Ser Leu Leu Ala Asp Thr 85 90 95 gcg att ttg ttc atc ggc ctc acc tta atg cac ggt tcg ccg ctg att 336 Ala Ile Leu Phe Ile Gly Leu Thr Leu Met His Gly Ser Pro Leu Ile 100 105 110 gtg aag ctg atc gac cag gtc gtc gtc atc gtg ctc aat tat ggc ttc 384 Val Lys Leu Ile Asp Gln Val Val Val Ile Val Leu Asn Tyr Gly Phe 115 120 125 agc aaa cta att ttc gct taa 405 Ser Lys Leu Ile Phe Ala 130 7 134 PRT Lactobacillus brevis 7 Met Glu Lys Pro Ile Thr Thr Leu Tyr Ser Lys Tyr Asp Thr Ala Leu 1 5 10 15 Arg Tyr Leu Ile Val Gly Gly Leu Thr Thr Gly Ile Asn Val Val Leu 20 25 30 Phe Phe Gly Leu Thr His Leu Ala Met Pro Trp Phe Trp Ala Asn Ile 35 40 45 Ile Ala Trp Val Leu Ser Val Leu Phe Ala Phe Ile Ala Asn Lys Lys 50 55 60 Val Val Phe Asn Ser Ala Asp Met Thr Phe Arg Thr Val Val Lys Glu 65 70 75 80 Gly Ala Ser Phe Phe Thr Leu Arg Gly Ala Ser Leu Leu Ala Asp Thr 85 90 95 Ala Ile Leu Phe Ile Gly Leu Thr Leu Met His Gly Ser Pro Leu Ile 100 105 110 Val Lys Leu Ile Asp Gln Val Val Val Ile Val Leu Asn Tyr Gly Phe 115 120 125 Ser Lys Leu Ile Phe Ala 130 8 10 DNA Lactobacillus brevis 8 actgcgacca 10 9 10 DNA Lactobacillus brevis 9 ggatcgtcgg 10 10 10 DNA Lactobacillus brevis 10 agtgcacacc 10 11 20 DNA Lactobacillus brevis 11 gtcagcgtgc cgacatcctg 20 12 20 DNA Lactobacillus brevis 12 tgtattcacc aatcaccccg 20 13 20 DNA Lactobacillus brevis 13 gccccgactt gaccatttgt 20 14 20 DNA Lactobacillus brevis 14 ttagcgggtg agcagcgagc 20 15 20 DNA Lactobacillus brevis 15 acagccttgc gctacctgat 20 16 20 DNA Lactobacillus brevis 16 ttcacaatca gcggcgaacc 20 17 20 DNA Lactobacillus brevis 17 tgagttttag taatattagt 20 18 20 DNA Lactobacillus brevis 18 agccaagctt gatgccggca 20 19 20 DNA Lactobacillus brevis 19 aaagtaactt agaaaaacaa 20 20 20 DNA Lactobacillus brevis 20 atgatctacg gactttacct 20 21 25 DNA Lactobacillus brevis 21 tcaatatgaa aaactagtcg agcag 25 22 24 DNA Lactobacillus brevis 22 ttatggacgt taacatagtc agca 24 23 25 DNA Lactobacillus brevis 23 ggaagatgct cagtgggacc gaatc 25 24 23 DNA Lactobacillus brevis 24 gccttttgat gcgctcgaac gat 23 25 25 DNA Lactobacillus brevis 25 tcacagaaag attaagtcgg caaca 25 26 24 DNA Lactobacillus brevis 26 tctaattctt tggcgctaac cgtc 24 27 25 DNA Lactobacillus brevis 27 aattgaaagt aagttgcgaa agaaa 25 28 20 DNA Lactobacillus brevis 28 ggcgaaccgt gaacaaatag 20 29 25 DNA Lactobacillus brevis 29 tacaattagt aagacaacag ggatt 25 30 20 DNA Lactobacillus brevis 30 tcaggcaatt cttgttcatc 20 31 16 DNA Lactobacillus brevis 31 ttactggccg ttgaag 16 32 15 DNA Lactobacillus brevis 32 tgagcttgcc gatgt 15 33 20 DNA Lactobacillus brevis 33 gatgccgacc tccaagatga 20 34 18 DNA Lactobacillus brevis 34 catgcccacc gccagtag 18 35 17 DNA Lactobacillus brevis 35 ccgacttccg cctgatg 17 36 20 DNA Lactobacillus brevis 36 ggtgagcttg ccgatgtatt 20 37 16 DNA Lactobacillus brevis 37 cgcgcaaacc gtcctc 16 38 20 DNA Lactobacillus brevis 38 agcttgccga tgtattcacc 20 39 25 DNA Lactobacillus brevis 39 tcgccggcat gagtgaagtc gtgaa 25 40 24 DNA Lactobacillus brevis 40 cggcgcaatc gttaggctgg tgat 24 41 16 DNA Lactobacillus brevis 41 gcgctgttgg tggtag 16 42 15 DNA Lactobacillus brevis 42 ctgggctgct tgatg 15 43 16 DNA Lactobacillus brevis 43 ttactggcga tgctga 16 44 16 DNA Lactobacillus brevis 44 cttggggatg gttttc 16 45 16 DNA Lactobacillus brevis 45 gtcgccgttt gccatc 16 46 16 DNA Lactobacillus brevis 46 cgcttgggga tggttt 16 47 20 DNA Lactobacillus brevis 47 tcgtggcctt cggtttcttt 20 48 19 DNA Lactobacillus brevis 48 cgcttgggga tggttttca 19 49 25 DNA Lactobacillus brevis 49 catccggttg tgggtagtga agtta 25 50 23 DNA Lactobacillus brevis 50 gtggcaaggt tagtgagggt gac 23 51 15 DNA Lactobacillus brevis 51 gccttgcgct acctg 15 52 15 DNA Lactobacillus brevis 52 gtgtccgcca gcagt 15 53 20 DNA Lactobacillus brevis 53 tcttcggcct gactcacctc 20 54 19 DNA Lactobacillus brevis 54 gcacgatgac gacgacctg 19 55 20 DNA Lactobacillus brevis 55 ctcgcgatgc cgtggttctg 20 56 19 DNA Lactobacillus brevis 56 ccgtgtccgc cagcagtga 19 57 24 DNA Lactobacillus brevis 57 ccttgcgcta cctgattgtt ggag 24 58 25 DNA Lactobacillus brevis 58 cataattgag cacgatgacg acgac 25 59 16 DNA Lactobacillus brevis 59 tgaatgggcg agtgat 16 60 15 DNA Lactobacillus brevis 60 ggcagccaaa tcgtg 15 61 15 DNA Lactobacillus brevis 61 gccagtgccg cttat 15 62 20 DNA Lactobacillus brevis 62 ttctttctgt tcggattcac 20 63 20 DNA Lactobacillus brevis 63 gtgaatccga acagaaagaa 20 64 15 DNA Lactobacillus brevis 64 acagccagcg aatgc 15 65 20 DNA Lactobacillus brevis 65 gataaggaag gtcgccacta 20 66 17 DNA Lactobacillus brevis 66 gcagccaaat cgtgatg 17 67 25 DNA Lactobacillus brevis 67 aaaggacgaa gtgcgattgc cagtg 25 68 23 DNA Lactobacillus brevis 68 cgttcatcac agccagcgaa tgc 23 69 15 DNA Lactobacillus brevis 69 gcgacggtct ctgtt 15 70 17 DNA Lactobacillus brevis 70 gtttcttacc cgattgc 17 71 17 DNA Lactobacillus brevis 71 cgacggtctc tgttgaa 17 72 20 DNA Lactobacillus brevis 72 ccactaactt gcctcacaat 20 73 20 DNA Lactobacillus brevis 73 gctatcgctg tctttttgaa 20 74 19 DNA Lactobacillus brevis 74 aatttttcgc tcctttggt 19 75 25 DNA Lactobacillus brevis 75 tggcagacgt caagtatttg ttcac 25 76 25 DNA Lactobacillus brevis 76 tcaatttttc gctcctttgg tatga 25 77 24 DNA Lactobacillus brevis 77 gaaattcatc aagtcacgcc ctat 24 78 25 DNA Lactobacillus brevis 78 tctcaatttt tcgctccttt ggtat 25 79 9901 DNA Lactobacillus brevis 79 atcacttgct ggttgctttc tagatgaatt ttcggaacgc taaacattgg aatccctctc 60 ttcgattgat ggttgcggta acttcaatct aacagattgg gtttcttttt ttatcaccca 120 acaggtgaaa agtgaaaaca ttagacaacg ttgatactac agtaactgtc gtaaattttg 180 ggatttatat gaataattcc aggtttagct tagcaaacaa aggattcctg taattgcatg 240 ttgtatttga cttgtatcag gcaattcttg ttcatcaatt aggtaattaa aactacccag 300 aataagtgaa gttaataatg acatttcaaa cttatcaggt ttttcctcca gcatacttac 360 aagattgttt cttaaaaccc tttttatctc actactcaaa tctttgctat ccaattgaat 420 agagcgcaat gctaaaatct tttcacgttg tttgattaaa agatttttta tatcaggtgc 480 caatatggat gtaatgctca aaaagttttg attttgttta tttaaattac ttcttttctt 540 caatactgaa tcataagtcg acacaaaatc ctgaatcatt ttcgcggcca agtgatactt 600 atcctgatag tgcctataaa atgtctgacg attgatcaag gctttattag agatatcaat 660 taccgataca ttattaaatc cctgttgtct tactaattgt ataaaagaat tttcaattaa 720 catttctgtt ctcttatttc ttaaatcagt cataagttcc tccttttctc caaacatcag 780 tatagatcca tatgtaactt aagtatacac aaataacatt ttgtatgcct aagtgacgtt 840 tcaaaaaaag ttgtctatgt ctttccaaaa taatgtccac tattattagt acttagctta 900 aaagggagga atatcagatg ttcgatgtaa ttcgtagtaa aagatattgg ttagcattat 960 tgcttgttgg agcaattatt ggaatagttt catttgcctt cattggcata cgtaactctg 1020 tcaaagtaaa acaaattcct gtagcacttg tcaacgaaga caaaggagct ctcagcaata 1080 aaattgaaag taagttgcga aagaaattca atggaaaaga ttcaaaaatc aaatgggtat 1140 ctccacaaaa agatggtttt aatgatcaaa agtattatgg ggctttcatt attagatcag 1200 gattttcaaa agagttacag cagcaaaatg aatcgctaaa ggcccaaatt attagccaaa 1260 agctcactac tcttcaaaaa aaggagaaat taccagattc tgcaaaatca aaattgcttc 1320 aagctaaatt taaatcacaa ttgcttcaag ctaaatttgt tacacagaaa cccgttcacc 1380 ctgcacagat taagatcagt attaatcaag gaatgaatgc tcaaatatcg caattgctat 1440 cccaagcact tcctaagatt gcaaatgcgc tttcatcacg aattagcgca cagcaacaaa 1500 gtgttcttag caaaaataag attaatttat ccgcaaaatc ttgggatttg gtttcgaccc 1560 ctattagtgt atctactcat gagtctaata aaattgaaaa gaacacggtt aatggcacag 1620 cacccatgct tctagtggca ttggcgtggt ttagcgcttt aattccctct cttattttat 1680 ggcgcgaaca cacaaaaaga agcgcttcaa aatttttaaa tgctacaaca ataactagtc 1740 aactaattac cggtttggta gcaagtattc tttcagcaac agttgggttc ttatttgtta 1800 atgtatgctt taacctaaca attccaaacc caattaactt tatcggatta atgtctatta 1860 gtatctttgt cttttatctt attataacgt gtgtcctgga ttggttggga tttgctttct 1920 acccattact actcgtagtc tggctcctag caatttccgt gatatcttat gcaccggaaa 1980 cccttgatcc tttgtaccga aagggaattt acagttgggt tccaatgcga tttagcatgc 2040 aaacactaac aaatactttg tatttccata atggatcgag taccaccatg tcatcattat 2100 tagtcttgtt aataattgga tttgtcgctg ctatcttgat gtatagttca ggatacttaa 2160 aacactattt gttcacggtt cgcccacacc gcaaaattaa ataattaaac aaaacatgaa 2220 acccaattat tcatatcatt aaaaatagcc aatcaattga ttgactattt tttgatgatc 2280 catttagcaa caattccctc aaaaaaaaag agagtgtgaa atattttgtg taaatgtata 2340 taaaaattct gaattgtata gaggcagaga atgttcttga ggtataccct attggctaat 2400 cacacataaa agctttctta tatcattatt tttttggcag gtactgtgta ggtggaaatg 2460 aacgaatgct ttaatagttt tgtgtcacaa aataaatttt ggagattttc agcactccag 2520 tagttcaatg attcctggca tattccatca gcaagtttat atattgcgtc ttttaacaat 2580 tggagcttac tttcagaaac tacatgaact ttaaagttgg tatgtgcatc tgaagcaaag 2640 atgatagagg ggtggaaaat aatggaatag aaagtttgac tctgatattt ttccgcaaac 2700 caattcgaag atgtagttat ttgttctaca tcacctttaa agatttcatc actagctcgt 2760 cgatttttat cttctatgac aatctgtgtg ctttgatcca gccaaagatt atcaggaccg 2820 ccttctttga tagccgtttc tgcctcaggc tgactagaat caaatcctaa taattctcct 2880 aatctttgaa tgcttgctct aaagtttgtt tcgtcggcat agtttgtata aagcaggtca 2940 tcattaatag ctcgaatatg tgtagctaaa tcattgctat cttcgaagtc atactttttt 3000 atatatggta gattgcaaga attaaccgaa cactgcgaac tgctgaaaaa cctttcgtgg 3060 tgattgccaa ttgagtgttt tcattggtct tgcattaatc caatgattaa tttgatctaa 3120 ttctttggcg ctaaccgtct caatttttcg ctcctttggt atgaatcgcc gaacgtatcg 3180 gtttaatatc tcattactcc cacgttcttc tggtgagtat gggtgtgcaa agtaaactgg 3240 aatgcctagt ttttgctcga tttcatcata ttttacaaac tctcgtcctc gatcaacagt 3300 gattgactta gcattctcga tcccttgaaa aaaatgaatt aatactggtg tcactgcctg 3360 actattgcga ccactaactt gcctcacaat atgttgccga cttaatcttt ctgtgattgt 3420 gacaagcact tcaccacgtt tcttacccga ttgcatcgta tcaacttcaa agtgaccaaa 3480 atcctgccgt gcttgaacac ttttgggtct tgattcaatt gaacggccgt gaacaaatac 3540 ttgacgtctg ccatcagact gacgtttttg acgaatacct ttgtcaggta aatcagctaa 3600 tgacagtttg agtcggccag cattgagcca attataaatc gttttgaaag gtaaccctaa 3660 aacatgagca gcagtttctg gtgaccactt taaaatgcca atgtgctcat tcaaaaagac 3720 agcgatagct ggtgttagtg tgtgatgacg gccacgtaga tgacgttttt tcaatgccaa 3780 tgcatgagca atatcagctt tatagggcgt gacttgatga atttcaacag agaccgtcgc 3840 aggtgatcgg ttaataaaac gcgcgatggc acgaatcgaa taattcaatt ccagcaaagt 3900 ttgaatgaca gtacgttctt gagatgataa actagtcatg agtcgcagtt cctttatggt 3960 tgttttggac aattaccatt aaaggcacaa ttcacatagg gagaagacca ttgaaatcca 4020 ccattttact agtcgaagac gaggcgggct tagccgactc actcaaaacc gaatttgagc 4080 tcgagaactt caatgtgttc tgggccaatg acggcctaat cgcgctggac atgttccggc 4140 aaaacgaggc ccagattgat ttaatcattt tggactggat gctgccgcac attcaagatc 4200 aagaaaccca gctgattact gtattgattg gcattggctg gatcgcgatg gtggcgatat 4260 cgcgggtgta tctgcgcgac cactacctct ctgatgtgct cgccagtgtc tgcttagcta 4320 gccgctggtg gttgctggtc acacctgcgg aagcctttat tcaagctaaa atgcggcagt 4380 ttttaccgga agggatgttg aaatcatggc cacatcaaaa ttaacgattg ttgttcctgc 4440 ttacaatgaa gaagaggtgc tgacgtcctc ggtgcaaaaa ttactggccg ttgaagacca 4500 aatcgccgcg caaaccgtcc tcggtcagcg tgccgacatc ctgatcgtcg atgatggttc 4560 catggatcac acctgggaca tcatcgaaaa actgcacgcc atgaattctc gcgtgcgcgg 4620 actgcgtttt tcccgcaact tcggccacca gtcggcgctg atcgccggca tgagtgaagt 4680 cgtgaaaacc gccgatatga ttgtcaccat cgatgccgac ctccaagatg atcccgacaa 4740 aatcggcgac atggtggatg cctatgcgga tggcgccgac atcgtctacg gcgtccggaa 4800 caaccgggaa accgacagct ggttcaagcg caccacggcc caaggctact acaagacact 4860 caagctgctg ggcgtcgaac tcgtgcccaa tcacgccgac ttccgcctga tgtccaagcg 4920 cgccgttgaa accttcctgc agtatccaga acgcaacatt ttcattcgcg gcctgattcc 4980 taagctcggc ttcaaaactg ccgaagtctt ctacaagcgc acaccgcgca tggccggcga 5040 atccaagtac ccgctgaaaa agatgctggc ttttgcctgg gacggcatca ccagcctaac 5100 gattgcgccg gtgcggctca ttctcattct gggtaccttg tcttgcctac tggcggtggg 5160 catggtggtt tacgccattg tcatgaaaat gctggggctc accgtgcacg gctggtcgtt 5220 gttgatggtg tcgctgtggt tcgttggcgg catccaaatg atcagcctcg gggtgattgg 5280 tgaatacatc ggcaagctca ccaccgaagt taaacatcgc ccgcgctaca cggtgcaaac 5340 gattctggat tgaggtgagg gtatgaaaaa gattaaaccc gcctttctgc cagcaatttt 5400 aatcctctgc ctgctcattg gcagcatcgg taacctgacc agtgtgctcg gggtgccggc 5460 gctgttggtg gtagtgctcc tgggcgcggg gctttacttc ggggcgcccc gacttgacca 5520 tttgtctaca aggcagctgc gctggggcat tggccttggc ttactggcga tgctgattgc 5580 ccaggtggtc gtgttgcacg tgatgcccaa caccgtttac cacgatccgt accgggtact 5640 gtcgcaagcc gaccagctcg ccgccggcca catgacctgg gatatcacct acttctggcg 5700 ctacgccaat aacgtgccgc tggcttatct gctctccctg tggttgcggc tgacgcaact 5760 ggtgggctta agcaccaatc tttcggtgca cctgctgagt atcttggtgt tggacagctt 5820 tattgccctg gcgctgcata cgatttggca gctcagccag cgcgccagcc tgctggtcgt 5880 ggccttcggt ttctttgcct tgtcgccgtt tgcctacacc tactacctgc aagtctttta 5940 ctccgactta ccgacgatgc tggtgctgct catcatcata cgcagcctgc tgaactggtc 6000 gcagaaaaca tcgcgccagc gctggtttgc cggcagcgga ctagttgttg ccgtgatgct 6060 cggcgccatg ctcaagccta atctggtggt cttgttgcca gctctgctga ttgtcggcct 6120 gattctggcc cgtcagcacc tctggcgaca agccaaactg accctgccca tcctcttgat 6180 tgtgctgggc ttcgggctga gtctgccggc gaccaaagtc tttgacgtgg cagccaatta 6240 tcaaccccgc accgcctttt cgttcccggc gacccactgg atcttgatgg gctacaacca 6300 gcacagcaac ggcggctact ccggcaagga tgtcggacgt gccatcaagc agcccagcca 6360 agccgaccgc cagcggtaca atttgaaaac catccccaag cgcatcaaaa ctctcggggt 6420 ggttggcgtc atccggttgt gggtagtgaa gttaggcatc gtgctcaatg tccaaggcat 6480 tcagcgctgg tacaacggcg gcttccgcgc cgcgcctagt tggtacagta atcatgctgg 6540 cttctatcag ggactgaccg tgattggcta tgtggccgcg accctgctca tgtggggcgc 6600 actgatgctg aagctcttgc ggtggcggcc agatctgacc gacccgcatc aaatccttgc 6660 actgctggcg gtgaccactg cccttggcta cctggctttc cacaccctac tgtgggaagt 6720 tgaaccgcgc tatggtcaag ccattttgcc gctgctctgg gtggctttgg cggccatccc 6780 gcgtcaggcc agccagtcgc gtccccgctg ggcgaaccaa gctagcctcc tcaatggcgc 6840 cactgcttca ctcgtcgcct ttggggccgc tggtgtgctt ggcgctcagc tgccacaaaa 6900 gcaagtgatt gccgcccagc gcagtcagct atccgtgcag tatcacgcca agcccaagac 6960 cgtgacgcca ggcaccgtgc tggcagaggt ggtcgatgtg aacgcgccag cgaactattt 7020 ttccgttcag attcatgctg gtagtcaggt gcaagtcacc ctcactaacc ttgccaccgg 7080 gcaacattat cggttaacga tggctggcag tgtggcccgc ctgcaccacc agctcgccgc 7140 tgggcaatat cggattaccg ttcaaaacct caccacccgc ggccagcagg tcgatgtgac 7200 ccacacctac cattatcagc tcgctgctca cccgctaacg gtgaacggcc aatcgcagcc 7260 caccgcctcg ttgatttata cctgcatgca gcgctgagaa aggagccttt ttatggaaaa 7320 accaatcact accctttaca gcaaatacga tacagccttg cgctacctga ttgttggagg 7380 cctcaccacc ggcattaatg tggtgctgtt cttcggcctg actcacctcg cgatgccgtg 7440 gttctgggcg aacattatcg cctgggtcct cagcgtgctg tttgccttca ttgccaacaa 7500 gaaagtcgtg ttcaactccg ccgacatgac cttccggact gtggtcaaag aaggcgccag 7560 cttcttcacc ttgcgcggcg cgtcactgct ggcggacacg gcgattttgt tcatcggcct 7620 caccttaatg cacggttcgc cgctgattgt gaagctgatc gaccaggtcg tcgtcatcgt 7680 gctcaattat ggcttcagca aactaatttt cgcttaacgt aaaaatggtc ccagcagtgg 7740 aaactgccga gaccattttg cttggctagc caagcttgat gccggcatcc gccagcgctg 7800 cgtcgaccac gttcatcaca gccagcgaat gcgccatccg ctgcttggcg gcagccaaat 7860 cgtgatgcgc aatcatcgtt tcgaacgcga cgaactcttc gtacatgcgg tgccggtcag 7920 ctaccatacc ttgatcgaca aaattttctt gcacatattt tgtcagtaat tctttctgtt 7980 cggattcact taattctacc ggtaaagcca cgttaaactc ttttgcatac cgtgagtttg 8040 atttacgatc tttcttttca acttcattcc acaactgctc tcgatcactc gcccattcag 8100 gtgaattttt tggcgtcaaa ataaagcttt ctggcatgat cgatcgggca taaaaatagt 8160 ggcgaccttc cttatcatca aatagctttt caccacttcg ataagcggca ctggcaatcg 8220 cacttcgtcc tttaccagca ctaatattac taaaactcat gtggaatatt gccatgtcag 8280 tcaccgtctt cctttgattc tatgggtttt gaccttgttg tcgccatcgg cgacttctaa 8340 tacaggattt tagcacgatt taccccacaa gaatttagtg gggtataagt gcgcattgac 8400 ctcgtttcac tcggccttct gattctctta actttaactt agtgtatgcc ttccgcttcg 8460 ctatttcaaa ttttatactt tgacttaacg tttcttatat gatatagttt cggtgattat 8520 ttctaatatg atttgaggga tcgttatgtc tcaaagtaac ttagaaaaac aagaagctaa 8580 attaaaagcc cttaatcaaa aaattaagga cgaaaaaaat aatattgaac aacggatagg 8640 taaacaaatc atcagtcaag ccaatttaga ttatgctaat ttgtctaacg atcagattaa 8700 gattttagcc aagcaatttt ctgaattttt aaaggtaaag tccgtagatc attagccata 8760 cgagatgggg aaattccatg tctaatcaat atgaaaaact agtcgagcag caagcgcgtt 8820 taaaacaaaa aattgagcgg gaagatttta aattacggca atctaaatac tatgaaaatc 8880 ggcaagcccg caaagcccgt tctcgccgat taattcaaaa aggggcttta ttagaaaagt 8940 actttcaagc taataacctc tcggtcgaac aaaccgaaga acttttaaaa acatttgctg 9000 actatgttaa cgtccataaa ccggataaat taaaaaacga tcaacctaat aactaggccg 9060 atcgttttta ttttcaggat tgttttttgg cttaatttct ggattgtgtt tagcaaagtc 9120 ttttaactgt ttttgaattc tttgtgtaag ctctgcttta ctttctttgt gcttcctttt 9180 acttgatcgc tgttgaatta tcttttgacc cttacctctt ctttttgatt ttaggtcaag 9240 cgtaaaatct gaaattttat tttgatctag cttacttaaa tgaccgattt ctttaaagtt 9300 taaaccggct ttgggaaagc gataaatatt accaagatcg acccaggaag gtttcttcaa 9360 cccagctgat tgccaatctt gaatcggata atattgttgc ttgatataag gagacttctt 9420 ttcatactga ctagggactg tctgaaaact aaaaattcag gtataatctg aggaaaaacg 9480 aatcgaggca ttccgatgac aacacctaaa cgatacgaac tggaagatgc tcagtgggac 9540 cgaatcaaag gatacttccc gccataccgg actggccgtc catcaagcct agacaaccgt 9600 accgccctca acgctatcct ctggctcatg cgcagcgggg ctccttggcg tgatctacct 9660 gaacgctatg gctcttggaa aacggtgtat agtcgcttcc gagcctgggt aagttcaggc 9720 ttgttcgaac aggtttttct cgaattgatt gacgatctcg acatggaaaa cttgagctta 9780 gattcaacga tcgttcgagc gcatcaaaag gccactgggg caaaaaaaac gccgaatgta 9840 tggtcgaaaa tcaagctatt ggactaagtc gaggtggccg aacgaccaag attcacgcac 9900 t 9901 

1. A probe for use in detecting beer-spoilage lactic acid bacteria, which comprises a nucleotide sequence consisting of at least 15 nucleotides that hybridizes with the nucleotide sequence of SEQ ID NO: 1 or the complementary sequence thereof.
 2. The probe according to claim 1, wherein the nucleotide sequence comprises at least 15 contiguous nucleotides of the nucleotide sequence of SEQ ID NO: 1 or the complementary sequence thereof.
 3. The probe according to claim 1, wherein the nucleotide sequence comprises at least 15 contiguous nucleotides from position 2818 to 8056 of the nucleotide sequence of SEQ ID NO: 1 or the complementary nucleotide sequence thereof.
 4. The probe according to claim 1, wherein the nucleotide sequence comprises at least 15 contiguous nucleotides from position 4202 to 7513 of the nucleotide sequence of SEQ ID NO: 1 or the complementary sequence thereof.
 5. The probe according to claim 1, wherein the nucleotide sequence comprises at least 15 contiguous nucleotides of the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6 or the complementary sequence thereof.
 6. The probe according to claim 1, wherein the nucleotide sequence comprises at least 20 nucleotides.
 7. The probe according to claim 1, wherein the nucleotide sequence is selected from the nucleotide sequences of SEQ ID NO: 8 through SEQ ID NO: 78 or the complementary sequences thereof.
 8. The probe according to claim 1, which is labeled.
 9. A method of detecting beer-spoilage lactic acid bacteria, which comprises the steps of hybridizing the polynucleotide probe of any one of claims 1 to 8 with a polynucleotide in a sample and then detecting a hybridization complex.
 10. The method according to claim 9, wherein the sample is a colony of bacteria obtained from beer.
 11. A polynucleotide primer for use in detecting beer-spoilage lactic acid bacteria by nucleic acid amplification reaction, which comprises a nucleotide sequence consisting of at least 15 nucleotides that hybridizes with the nucleotide sequence of SEQ ID NO: 1 or the complementary sequence thereof.
 12. The primer according to claim 11, wherein the nucleotide sequence comprises at least 0.15 contiguous nucleotides of the nucleotide sequence of SEQ ID NO: 1 or the complementary nucleotide sequence thereof.
 13. A primer pair for use in detecting beer-spoilage lactic acid bacteria, comprising two kinds of primers of claim 11, wherein the primer pair can amplify a genomic sequence specific to the beer-spoilage lactic acid bacteria by a nucleic acid amplification method.
 14. The primer pair according to claim 13, wherein one primer comprises at least 15 contiguous nucleotides of the nucleotide sequence of SEQ ID NO: 1 and the other primer comprises at least 15 contiguous nucleotides of the nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO:
 1. 15. The primer pair according to claim 13, wherein one primer comprises at least 15 contiguous nucleotides of the nucleotide sequence from position 2818 to position 8056 of SEQ ID NO: 1 and the other primer comprises at least 15 contiguous nucleotides of the sequence complementary to the nucleotide sequence from position 2818 to position 8056 of the nucleotide sequence of SEQ ID NO:
 1. 16. The primer pair according to claim 13, wherein one primer comprises at least 15 contiguous nucleotides of the nucleotide sequence from position 4202 to position 7513 of SEQ ID NO: 1 and the other primer comprises at least 15 contiguous nucleotides of the sequence complementary to the nucleotide sequence from position 4202 to position 7513 of the nucleotide sequence of SEQ ID NO:
 1. 17. The primer pair according to claim 13, wherein one primer comprises at least 15 contiguous nucleotides of the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6 and the other primer comprises at least 15 contiguous nucleotides of the sequence complementary to the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO:
 6. 18. The primer pair according to claim 13, wherein the contiguous nucleotides are at least 20 contiguous nucleotides.
 19. The primer pair according to claim 13, wherein the two kinds of primers are selected from the nucleotide sequences of SEQ ID NO: 8 through SEQ ID NO: 78 or the complementary sequences thereof.
 20. A method of detecting beer-spoilage lactic acid bacteria, which comprises the steps of amplifying a polynucleotide in a sample using the primer pair of claim 13 by nucleic acid amplification reaction and then detecting the amplified polynucleotide.
 21. The method according to claim 20, wherein the sample is a colony of bacteria obtained from beer.
 22. A protein comprising the amino acid sequence of SEQ ID NO:
 3. 23. A protein comprising the amino acid sequence of SEQ ID NO:
 5. 24. A protein comprising the amino acid sequence of SEQ ID NO:
 7. 25. A polynucleotide encoding the amino acid sequence of claim
 22. 26. A polynucleotide encoding the amino acid sequence of claim
 23. 27. A polynucleotide encoding the amino acid sequence of claim
 24. 28. A recombinant vector carrying the polynucleotide of claim
 25. 29. A recombinant vector carrying the polynucleotide of claim
 26. 30. A recombinant vector carrying the polynucleotide of claim
 27. 31. A host comprising the recombinant vector of claim
 28. 32. A host comprising the recombinant vector of claim
 29. 33. A host comprising the recombinant vector of claim
 30. 34. A method for producing a protein, which comprises the steps of culturing a host comprising a recombinant vector carrying the polynucleotide of claim 25 and collecting an expression product of said polynucleotide from the culture.
 35. A method for producing a protein, which comprises the steps of culturing a host comprising a recombinant vector carrying the polynucleotide of claim 26 and collecting an expression product of said polynucleotide from the culture.
 36. A method for producing a protein, which comprises the steps of culturing a host comprising a recombinant vector carrying the polynucleotide of claim 27 and collecting the protein, that is, an expression product of said polynucleotide, from the culture.
 37. A protein which is obtainable by the steps of culturing a host cell comprising a recombinant vector carrying the polynucleotide of claim 25 and collecting the protein that is an expression product of said polynucleotide, from the culture.
 38. A protein which is obtainable by the steps of culturing a host cell comprising a recombinant vector carrying the polynucleotide of claim 26 and collecting the protein that is an expression product of said polynucleotide, from the culture.
 39. A protein which is obtainable by the steps of culturing a host cell comprising a recombinant vector carrying the polynucleotide of claim 27 and collecting the protein that is an expression product of said polynucleotide, from the culture.
 40. An antibody against the protein of any one of claims 22, 23, 24, 37, 38, and
 39. 41. The antibody according to claim 40, wherein the antibody is a polyclonal antibody.
 42. The antibody according to claim 40, wherein the antibody is a monoclonal antibody.
 43. A method of detecting beer-spoilage lactic acid bacteria, which comprises the steps of reacting the antibody of claim 40 with a sample and detecting antigen-antibody reaction between the antibody and the antigen.
 44. The method according to claim 43, wherein the sample is a colony of bacteria obtained from beer. 